Alpha-4 beta-7 heterodimer specific antagonist antibody

ABSTRACT

There are disclosed alpha4beta7 heterodimer-specific antigen binding proteins, nucleic acids encoding them, and methods of making and using them.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. 119(e) of U.S.patent application No. 61/162,154, filed Mar. 20, 2009 and U.S. patentapplication No. 61/306,829, filed Feb. 22, 2010, which are incorporatedherein by reference.

REFERENCE TO THE SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitledA-1459-US-NP_Seq_Listing.txt., created Mar. 16, 2010, which is 84.0 KBin size. The information in the electronic format of the SequenceListing is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

This application provides compositions and methods relating toalpha4beta7 heterodimer-specific antigen binding proteins.

BACKGROUND

Integrins are heterodimeric Type I transmembrane proteins formed of twosubunits (one alpha subunit and one beta subunit), and mediate manydifferent cell-cell and cell-extracellular matrix interactions.Functionally, integrins have been shown to be involved in diversebiological processes, including leukocyte migration and recirculationand the immune response. In mammals, there are 18 known alpha subunitsand eight known beta subunits, which combine to form 24 distinctintegrins. Ligand specificity is determined in large part by theparticular combinations of alpha and beta subunits expressed, whileaffinity for ligand is modulated by integrin conformational changes andis divalent-cation dependent.

The ligands for integrins form a structurally diverse group thatincludes extracellular matrix proteins such as collagens, fibronection,vitronectin and laminins; counter-receptors such as the cellularadhesion molecules (for example, vascular cellular adhesion molecule orVCAM), and plasma proteins. Numerous pathogenic microorganisms alsoutilize integrins to initiate infection or as sites for toxin binding.The structurally diverse ligands share an exposed glutamic or asparticacid residue, usually present in an extended, flexible loop, which isimportant for recognition by integrins.

The alpha4 integrins (alpha 4 partnered with either the beta1 or beta7subunit) play an important role in the immune system. Alpha4beta1 isexpressed on lymphocytes and myeloid cells; it appears to be the majorbinding partner for vascular cell adhesion molecule (VCAM). VCAM isubiquitously expressed on vascular endothelium, is up regulated duringinflammation, and binds alpha4beta7 as well as alpha4beta1 (albeitweakly to alpha4beta7). Though also detected on d peripheral T cells, Bcells, NK cells and eosinphils, alpha4beta7 is most highly expressed ona subpopulation of CD4+CD45RA-memory T cells which has been shown topreferentially home to the gut. The primary ligand for the alpha4beta7heterodimer is mucosal addressin cell adhesion molecule 1 (MAdCAM-1 orMAdCAM), which is expressed in gut endothelium.

In addition to pairing with the alpha4 chain, the beta7 subunit alsopartners with alphaE to form alphaEbeta7, which is primarily expressedon intraepithelial lymphocytes (IEL) in intestine, lung andgenitourinary tract. AlphaEbeta7 is also expressed on dendritic cells inthe gut. The alphaEbeta7 heterodimer binds to E-cadherin, which isexpressed on epithelial cells. The IEL cells are thought to provide amechanism for immunosurveillance within the epithelial compartment.

Antibodies that bind alpha4 and inhibit binding of alpha4beta1 to VCAM-1and fibronection mapped to a 52-amino acid region of alpha4, betweenresidues 152 and 203 (Schiffer et al., J. Biol. Chem. 270:14270; 1995).Tidswell et al. (J. Immuno 159:1497; 1997) identified domains of beta7that are important in binding to MAdCAM-1, utilizing a panel ofantibodies that bind beta7 in a mouse/human chimeric beta7 subunitapproach. They found that six of seven antibodies that inhibited bindingto MAdCAM-1 and E-cadherin mapped to a region comprising amino acids 176through 250, which appears to have homology to the metal-ion dependentadhesion site (MIDAS) of other integrin subunits. One of the antibodiesused by Tidswell et al. was an alpha4beta7 heterodimer specific antibodyreferred to as ACT-1.

The ACT-1 antibody was originally described by Lazarovitz et al. (J.Immunol. 133:1857; 1984) as an antibody developed by immunizing micewith human tetanus toxoid-specific T lymphocyte line from PBMC. Later itwas shown that ACT-1 binds to the alpha4beta7 heterodimer specifically(Schweighoffer et al., J. Immunol. 151:717, 1993). While ACT-1 does notbind murine alpha4beta7, it does bind alpha4beta7 from least somenon-human primate species, and has been shown to attenuate spontaneouscolitis in captive cotton-top tamarins (Hesterberg et al.,Gastroenterology 111:1373; 1996)

ACT-1 has been humanized and evaluated as a human therapeutic inulcerative colitis (Feagan et al., N Engl J. Med. 352:2499; 2005), andrecently in Crohn's disease (Feagan et al, Clinical Gastroenterology andHepatology, 6:1370, 2008). Humanized ACT-1, also known as vedolizumab,is described in WO 98/06248 and U.S. Pat. No. 7,147,85, as well as WO07/061,679 and US 2007-0122404. Another humanized antibody, natalizumab(Tysabri®), has been used to treat Crohn's disease. Natalizumab is ahumanized version of an alpha4-specific murine antibody. Vedolizumab hasbeen shown to lead to a neutralizing anti-humanized antibody response ina portion of patients, and natalizumab has been associated withprogressive multifocal leukoencephalopathy (PML), a neurologicaldisorder that is associated with reactivation of prior infection with JCvirus in immunocompromised individuals. Accordingly, there is a need fora therapeutic agent that ameliorates these disadvantages whiledisrupting the alpha4beta7/MAdCAM-1 pathway.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides an isolated antigenbinding protein that specifically binds to human alpha4beta7 (i.e., analpha4beta7 heterodimer specific antigen binding protein). In anotheraspect of the invention, the antigen binding protein specifically bindsto the alpha4beta7 of a non-human primate, a cynomologous monkey, achimpanzee, a non-primate mammal, a rodent, a mouse, a rat, a hamster, aguinea pig, a cat, or a dog. In another embodiment, the isolated antigenbinding protein comprises a human antibody; a chimeric antibody; amonoclonal antibody; a recombinant antibody; an antigen-binding antibodyfragment; a single chain antibody; a diabody; a triabody; a tetrabody; aFab fragment; a F(ab′)₂ fragment; a domain antibody; an IgD antibody; anIgE antibody; an IgM antibody; an IgG1 antibody; an IgG2 antibody; anIgG3 antibody; an IgG4 antibody; or an IgG4 antibody having at least onemutation in a hinge region that alleviates a tendency to form intra-Hchain disulfide bond. In another aspect, the isolated antigen bindingprotein comprises a heavy chain constant region from one of theaforementioned antibodies; in another aspect, the constant region is apolypeptide comprising SEQ ID NO:72; a polypeptide at least 90%identical to SEQ ID NO:72; a polypeptide having an amino acid sequenceas set forth in SEQ ID NO:72 from which one, two, three, four or fiveN-terminal and/or C-terminal amino acids have been removed; or one ofthe afore-mentioned polypeptides which incorporates one or morepost-translational modifications. In one embodiment, the isolatedantigen binding protein comprises a kappa light chain constant region,in another it comprises a lambda light chain region. In one embodiment,the light chain constant region is a polypeptide comprising SEQ IDNO:70; a polypeptide at least 90% identical to SEQ ID NO:70; apolypeptide having an amino acid sequence as set forth in SEQ ID NO:70from which one, two, three, four or five N-terminal and/or C-terminalamino acids have been removed; or one of the afore-mentionedpolypeptides which incorporates one or more post-translationalmodifications

One embodiment of the present invention provides an alpha4beta7heterodimer specific antigen binding protein having a heavy chain and alight chain, each of which comprise one or more complementaritydetermining regions, or CDRs. In another aspect of the invention, theheavy chain variable region comprises CDR1, CDR2 and CDR3 and a lightchain variable region comprises CDR1, CDR2 and CDR3, wherein eachrespective CDR is selected from the group consisting of the light chainCDR1, CDR2 and CDR3 from SEQ ID NO:55, and the heavy chain CDR1, CDR2and CDR3 from SEQ ID NO:58; the light chain CDR1, CDR2 and CDR3 from SEQID NO:56, and the heavy chain CDR1, CDR2 and CDR3 from SEQ ID NO:59; andthe light chain CDR1, CDR2 and CDR3 from SEQ ID NO:57, and the heavychain CDR1, CDR2 and CDR3 from SEQ ID NO:60.

In another aspect of the invention, the heavy chain variable regionfurther comprises four framework regions (FRs) designated FR1, FR2, FR3and FR4, and the light chain variable region further comprises fourframework regions (FRs) designated FR1, FR2, FR3 and FR4. In one aspect,the FRs are selected from the same SEQ ID NO as the CDRs; in another,the FRs are selected from a different SEQ ID NO. In a furtherembodiment, the invention provides an alpha4beta7 heterodimer specificantigen binding protein wherein the light chain variable regioncomprises SEQ ID NO:55, and the heavy chain variable region comprisesSEQ ID NO:58; the light chain variable region comprises SEQ ID NO:56,and the heavy chain variable region comprises SEQ ID NO:59; or the lightchain variable region comprises SEQ ID NO:57, and the heavy chainvariable region comprises SEQ ID NO:60.

In another aspect of the invention, the present invention provides anisolated alpha4beta7 heterodimer specific antigen binding protein,having a heavy chain and a light chain, each of which comprise one ormore complementarity determining regions, or CDRs. In another aspect ofthe invention, the heavy chain variable region comprises CDR1, CDR2 andCDR3 and the light chain variable region comprises CDR1, CDR2 and CDR3.In one embodiment, the light chain CDRs are selected from the groupconsisting of a CDR1, CDR2 and CDR3 at least 90% identical to a CDR1,CDR2 and CDR3, respectively, of SEQ ID NO: 3; a CDR1, CDR2 and CDR3 atleast 90% identical to a CDR1, CDR2 and CDR3, respectively, of SEQ IDNO: 5; a CDR1, CDR2 and CDR3 at least 90% identical to a CDR1, CDR2 andCDR3, respectively, of SEQ ID NO: 7; a CDR1, CDR2 and CDR3 at least 90%identical to a CDR1, CDR2 and CDR3, respectively, of SEQ ID NO: 22; anda CDR1, CDR2 and CDR3 at least 90% identical to a CDR1, CDR2 and CDR3,respectively, of SEQ ID NO: 24; and the heavy chain variable CDR1, CDR2and CDR3 are from SEQ ID NO:58.

In another aspect of the invention, the heavy chain variable regionfurther comprises four framework regions (FRs) designated FR1, FR2, FR3and FR4, and the light chain variable region further comprises fourframework regions (FRs) designated FR1, FR2, FR3 and FR4. In one aspect,the FRs are selected from the same SEQ ID NO as the CDRs; in another,the FRs are selected from a different SEQ ID NO. In a furtherembodiment, the invention provides an alpha4beta7 heterodimer specificantigen binding protein wherein the light chain variable region isselected from the group consisting of a light chain variable region atleast 90% identical to SEQ ID NO:3; a light chain variable region atleast 90% identical to SEQ ID NO:5; a light chain variable region atleast 90% identical to SEQ ID NO:7; a light chain variable region atleast 90% identical to SEQ ID NO:22; and a light chain variable regionat least 90% identical to SEQ ID NO:24; and the heavy chain variableregion comprises SEQ ID NO:58.

Another aspect of the invention provides an isolated, alpha4beta7heterodimer specific antigen binding protein having a heavy chainvariable region comprising CDR1, CDR2 and CDR3 and a light chainvariable region comprising CDR1, CDR2 and CDR3, wherein the light chainCDR1, CDR2 and CDR3 are selected from the group consisting of a CDR1,CDR2 and CDR3 at least 90% identical to a CDR1, CDR2 and CDR3,respectively, of SEQ ID NO:12; a CDR1, CDR2 and CDR3 at least 90%identical to a CDR1, CDR2 and CDR3, respectively, of SEQ ID NO: 25; anda CDR1, CDR2 and CDR3 at least 90% identical to a CDR1, CDR2 and CDR3,respectively, of SEQ ID NO: 26; and the heavy chain CDR1, CDR2 and CDR3are selected from the group consisting of a CDR1, CDR2 and CDR3 at least90% identical to a CDR1, CDR2 and CDR3, respectively, of SEQ ID NO:41;and a CDR1, CDR2 and CDR3 at least 90% identical to a CDR1, CDR2 andCDR3, respectively, of SEQ ID NO:54. In one embodiment, the light chainvariable region is selected from the group consisting of variableregions that are at least 90% identical to any one of SEQ ID NOs: 12, 25and 26, and the heavy variable region is selected from the groupconsisting of variable regions that are at least 90% identical to anyone of SEQ ID NOs:41 and 54. In another aspect of the invention, theheavy chain variable region further comprises four framework regions(FRs) designated FR1, FR2, FR3 and FR4, and the light chain variableregion further comprises four framework regions (FRs) designated FR1,FR2, FR3 and FR4. In one aspect, the FRs are selected from the same SEQID NO as the CDRs; in another, the FRs are selected from a different SEQID NO.

In one embodiment, the invention provides an isolated, alpha4beta7heterodimer specific antigen binding protein having a heavy chainvariable region comprising CDR1, CDR2 and CDR3 and a light chainvariable region comprising CDR1, CDR2 and CDR3, wherein each respectiveCDR is at least 90% identical to a CDR selected from the groupconsisting of a light chain CDR1, CDR2 and CDR3 from SEQ ID NO:10, and aheavy chain CDR1, CDR2 and CDR3 from SEQ ID NO:38; a light chain CDR1,CDR2 and CDR3 from SEQ ID NO:2, and a heavy chain CDR1, CDR2 and CDR3from SEQ ID NO:30; a light chain CDR1, CDR2 and CDR3 from SEQ ID NO:20,and a heavy chain CDR1, CDR2 and CDR3 from SEQ ID NO:51; a light chainCDR1, CDR2 and CDR3 from SEQ ID NO:11, and a heavy chain CDR1, CDR2 andCDR3 from SEQ ID NO:39; a light chain CDR1, CDR2 and CDR3 from SEQ IDNO:13, and a heavy chain CDR1, CDR2 and CDR3 from SEQ ID NO:42; a lightchain CDR1, CDR2 and CDR3 from SEQ ID NO:17, and a heavy chain CDR1,CDR2 and CDR3 from SEQ ID NO:46; a light chain CDR1, CDR2 and CDR3 fromSEQ ID NO:8, and a heavy chain CDR1, CDR2 and CDR3 from SEQ ID NO:36; alight chain CDR1, CDR2 and CDR3 from SEQ ID NO:19, and a heavy chainCDR1, CDR2 and CDR3 from SEQ ID NO:49; a light chain CDR1, CDR2 and CDR3from SEQ ID NO:18, and a heavy chain CDR1, CDR2 and CDR3 from SEQ IDNO:47; a light chain CDR1, CDR2 and CDR3 from SEQ ID NO:21, and a heavychain CDR1, CDR2 and CDR3 from SEQ ID NO:52; a light chain CDR1, CDR2and CDR3 from SEQ ID NO:3, and a heavy chain CDR1, CDR2 and CDR3 fromSEQ ID NO:31; a light chain CDR1, CDR2 and CDR3 from SEQ ID NO:7, and aheavy chain CDR1, CDR2 and CDR3 from SEQ ID NO:35; a light chain CDR1,CDR2 and CDR3 from SEQ ID NO:6, and a heavy chain CDR1, CDR2 and CDR3from SEQ ID NO:34; a light chain CDR1, CDR2 and CDR3 from SEQ ID NO:1,and a heavy chain CDR1, CDR2 and CDR3 from SEQ ID NO:29; a light chainCDR1, CDR2 and CDR3 from SEQ ID NO:22, and a heavy chain CDR1, CDR2 andCDR3 from SEQ ID NO:50; a light chain CDR1, CDR2 and CDR3 from SEQ IDNO:24, and a heavy chain CDR1, CDR2 and CDR3 from SEQ ID NO:40; a lightchain CDR1, CDR2 and CDR3 from SEQ ID NO:9, and a heavy chain CDR1, CDR2and CDR3 from SEQ ID NO:37; a light chain CDR1, CDR2 and CDR3 from SEQID NO:4, and a heavy chain CDR1, CDR2 and CDR3 from SEQ ID NO:32; alight chain CDR1, CDR2 and CDR3 from SEQ ID NO:28, and a heavy chainCDR1, CDR2 and CDR3 from SEQ ID NO:53; a light chain CDR1, CDR2 and CDR3from SEQ ID NO:16, and a heavy chain CDR1, CDR2 and CDR3 from SEQ IDNO:45; a light chain CDR1, CDR2 and CDR3 from SEQ ID NO:15, and a heavychain CDR1, CDR2 and CDR3 from SEQ ID NO:44; a light chain CDR1, CDR2and CDR3 from SEQ ID NO:14, and a heavy chain CDR1, CDR2 and CDR3 fromSEQ ID NO:43; a light chain CDR1, CDR2 and CDR3 from SEQ ID NO:27, and aheavy chain CDR1, CDR2 and CDR3 from SEQ ID NO:43; a light chain CDR1,CDR2 and CDR3 from SEQ ID NO:5, and a heavy chain CDR1, CDR2 and CDR3from SEQ ID NO:33; a light chain CDR1, CDR2 and CDR3 from SEQ ID NO:12,and a heavy chain CDR1, CDR2 and CDR3 from SEQ ID NO:41; a light chainCDR1, CDR2 and CDR3 from SEQ ID NO:23, and a heavy chain CDR1, CDR2 andCDR3 from SEQ ID NO:48; a light chain CDR1, CDR2 and CDR3 from SEQ IDNO:25, and a heavy chain CDR1, CDR2 and CDR3 from SEQ ID NO:54; and alight chain CDR1, CDR2 and CDR3 from SEQ ID NO:26, and a heavy chainCDR1, CDR2 and CDR3 from SEQ ID NO:54. In another aspect, the heavychain and light chain CDRs are identical to the respective CDRs of therecited SEQ ID NOs. In one embodiment of the invention, the heavy chainvariable region further comprises four framework regions (FRs)designated FR1, FR2, FR3 and FR4, and the light chain variable regionfurther comprises four framework regions (FRs) designated FR1, FR2, FR3and FR4. In one aspect, the FRs are selected from the same SEQ ID NO asthe CDRs; in another, the FRs are selected from a different SEQ ID NO.

In another embodiment, an alpha4beta7 heterodimer specific antigenbinding protein comprises a light chain variable region and a heavychain variable region, wherein the light chain variable region is atleast 90% identical to SEQ ID NO:10, and the heavy chain variable regionis at least 90% identical to SEQ ID NO:38; the light chain variableregion is at least 90% identical to SEQ ID NO:2, and the heavy chainvariable region is at least 90% identical to SEQ ID NO:30; the lightchain variable region is at least 90% identical to SEQ ID NO:20, and theheavy chain variable region is at least 90% identical to SEQ ID NO:51;the light chain variable region is at least 90% identical to SEQ IDNO:11, and the heavy chain variable region is at least 90% identical toSEQ ID NO:39; the light chain variable region is at least 90% identicalto SEQ ID NO:13, and the heavy chain variable region is at least 90%identical to SEQ ID NO:42; the light chain variable region is at least90% identical to SEQ ID NO:17, and the heavy chain variable region is atleast 90% identical to SEQ ID NO:46; the light chain variable region isat least 90% identical to SEQ ID NO:8, and the heavy chain variableregion is at least 90% identical to SEQ ID NO:36; the light chainvariable region is at least 90% identical to SEQ ID NO:19, and the heavychain variable region is at least 90% identical to SEQ ID NO:49; thelight chain variable region is at least 90% identical to SEQ ID NO:18,and the heavy chain variable region is at least 90% identical to SEQ IDNO:47; the light chain variable region is at least 90% identical to SEQID NO:21, and the heavy chain variable region is at least 90% identicalto SEQ ID NO:52; the light chain variable region is at least 90%identical to SEQ ID NO:3, and the heavy chain variable region is atleast 90% identical to SEQ ID NO:31; the light chain variable region isat least 90% identical to SEQ ID NO:7, and the heavy chain variableregion is at least 90% identical to SEQ ID NO:35; the light chainvariable region is at least 90% identical to SEQ ID NO:6, and the heavychain variable region is at least 90% identical to SEQ ID NO:34; thelight chain variable region is at least 90% identical to SEQ ID NO:1,and the heavy chain variable region is at least 90% identical to SEQ IDNO:29; the light chain variable region is at least 90% identical to SEQID NO:22, and the heavy chain variable region is at least 90% identicalto SEQ ID NO:50; the light chain variable region is at least 90%identical to SEQ ID NO:24, and the heavy chain variable region is atleast 90% identical to SEQ ID NO:40; the light chain variable region isat least 90% identical to SEQ ID NO:9, and the heavy chain variableregion is at least 90% identical to SEQ ID NO:37; the light chainvariable region is at least 90% identical to SEQ ID NO:4, and the heavychain variable region is at least 90% identical to SEQ ID NO:32; thelight chain variable region is at least 90% identical to SEQ ID NO:28,and the heavy chain variable region is at least 90% identical to SEQ IDNO:53; the light chain variable region is at least 90% identical to SEQID NO:16, and the heavy chain variable region is at least 90% identicalto SEQ ID NO:45; the light chain variable region is at least 90%identical to SEQ ID NO:15, and the heavy chain variable region is atleast 90% identical to SEQ ID NO:44; the light chain variable region isat least 90% identical to SEQ ID NO:14, and the heavy chain variableregion is at least 90% identical to SEQ ID NO:43; the light chainvariable region is at least 90% identical to SEQ ID NO:27, and the heavychain variable region is at least 90% identical to SEQ ID NO:43; thelight chain variable region is at least 90% identical to SEQ ID NO:5,and the heavy chain variable region is at least 90% identical to SEQ IDNO:33; the light chain variable region is at least 90% identical to SEQID NO:12, and the heavy chain variable region is at least 90% identicalto SEQ ID NO:41; the light chain variable region is at least 90%identical to SEQ ID NO:23, and the heavy chain variable region is atleast 90% identical to SEQ ID NO:48; the light chain variable region isat least 90% identical to SEQ ID NO:25, and the heavy chain variableregion is at least 90% identical to SEQ ID NO:54; or the light chainvariable region is at least 90% identical to SEQ ID NO:26, and the heavychain variable region is at least 90% identical to SEQ ID NO:54. Inanother aspect, the heavy chain and light chain variable regions areidentical to the respective variable regions of the recited SEQ ID NOs.

One aspect of the invention provides an isolated, alpha4beta7heterodimer specific antigen binding protein having an EC50 of less than35 ng/ml in a CD4+ memory T cell binding assay; another provides anisolated, alpha4beta7 heterodimer specific antigen binding which has anEC50 of less than 10 ng/ml in a CD4+ memory T cell binding assay. Inanother embodiment, the invention provides an isolated, alpha4beta7heterodimer specific antigen binding protein having an IC50 in a MAdCAMcompetition assay of less than 30 ng/m; in another is provided anisolated, alpha4beta7 heterodimer specific antigen binding which has anIC50 of less than 10 ng/ml in a MAdCAM competition assay. One aspect ofthe invention provides an isolated, alpha4beta7 heterodimer specificantigen binding protein that binds an S250N mutant of alha4beta7.

In one aspect of the invention, the present invention provides nucleicacids encoding the aforementioned polypeptides. In another aspect of theinvention the nucleic acid is a vector. In another embodiment of theinvention, the invention provides host cells transformed or transfectedwith the inventive nucleic acids. In another aspect of the invention,there is provided a method of preparing a polypeptide comprisingincubating the host cells under conditions promoting expression of thepolypeptides and harvesting the polypeptides.

In another aspect, the present invention provides an isolated cell thatsecretes an antigen binding protein that binds alpha4beta7. In anotherembodiment, the cell is a hybridoma. In another embodiment, the presentinvention provides a method of making an antigen binding protein thatspecifically binds alpha4beta7 (i.e., human alpha4beta7), comprisingincubating said isolated cell under conditions that allow it to expresssaid antigen binding protein.

In one aspect, the present invention provides an isolated antigenbinding protein that specifically binds to an alpha4beta7 heterodimer.In another embodiment, the isolated antigen binding protein, when boundto a human alpha4beta7, inhibits binding of alpha4beta7 to MAdCAM-1.Accordingly, one embodiment of the invention provides a method ofinhibiting at least one activity of alpha4beta7, comprising contacting acell expressing alpha4beta7 with an alpha4beta7 heterodimer-specificantigen binding protein such that the activity is partially or fullyinhibited. In one aspect, such method is carried out in vivo. In oneaspect of the invention, the isolated antigen binding protein inhibitsadhesion of cells expressing alpha4beta7 to cells expressing MAdCAM-1.In yet another aspect of the invention, the isolated antigen bindingprotein inhibits trafficking of cells expressing alpha4beta7 to areas ortissues populated by cells expressing MAdCAM-1; in one example of suchan embodiment, the isolated antigen binding proteins inhibit traffickingof lymphocytes to the gut.

In another aspect, the present invention provides a pharmaceuticalcomposition comprising the antigen binding protein. In one embodiment,the present invention provides a method of treating a condition in asubject comprising administering the pharmaceutical composition to thesubject, wherein the condition is treatable by reducing the activity(partially or fully) of alpha4beta7 in the subject. In anotherembodiment, the subject is a human being. In another embodiment, thecondition is an inflammatory condition of the gastrointestinal system.Thus, there is provided a method of treating an individual afflictedwith a condition characterized by inappropriate trafficking of cellsexpressing alpha4beta7 to tissues comprising cells expressing MAdCAM,comprising administering to the individual an alpha4beta7 heterodimerspecific antigen binding protein in am amount sufficient to inhibit(partially or fully) the trafficking of cells expressing alpha4beta7 totissues comprising cells expressing MAdCAM. In one embodiment, thecondition is inflammatory bowel disease, for example, ulcerativecolitis, Crohn's disease, Celiac disease (nontropical Sprue),enteropathy associated with seronegative arthropathies, microscopic orcollagenous colitis, eosinophilic gastroenteritis, or pouchitisresulting after proctocolectomy and ileoanal anastomosis. In anotherembodiment, the condition is s pancreatitis, insulin-dependent diabetesmellitus, mastitis, cholecystitis, cholangitis, pericholangitis, chronicbronchitis, chronic sinusitis, asthma or graft versus host disease.

In another embodiment, the method further comprises administering to thesubject a second treatment. In another embodiment, the second treatmentis administered to the subject before and/or simultaneously with and/orafter the pharmaceutical composition is administered to the subject. Inanother embodiment, the second treatment comprises an anti-inflammatoryagent. In another embodiment, the second pharmaceutical compositioncomprises an agent selected from the group consisting of non-steroidalanti-inflammatory drugs, steroids, and immunomodulating agents. Inanother embodiment, the method comprises administering to the subject athird treatment.

In another aspect, the present invention provides a method of increasingthe longevity of a subject comprising administering to the subject thepharmaceutical composition. In another aspect, the present inventionprovides a method of decreasing alpha4beta7 activity in a subject inneed thereof comprising administering to the subject the pharmaceuticalcomposition. In another aspect, the present invention provides a methodof decreasing alpha4beta7-mediated trafficking (for example,alpha4beta7mediated gut homing) in a subject in need thereof comprisingadministering to the subject the pharmaceutical composition.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides compositions, kits, and methods relatingto molecules that bind to the integrin alpha4beta7 (“alpha4beta7”),including molecules that agonize or antagonize alpha4beta7, such asanti-alpha4beta7 antibodies, antibody fragments, and antibodyderivatives, e.g., antagonistic anti-alpha4beta7 antibodies, antibodyfragments, or antibody derivatives. Also provided are nucleic acids, andderivatives and fragments thereof, comprising a sequence of nucleotidesthat encodes all or a portion of a polypeptide that binds toalpha4beta7, e.g., a nucleic acid encoding all or part of ananti-alpha4beta7 antibody, antibody fragment, or antibody derivative,plasmids and vectors comprising such nucleic acids, and cells or celllines comprising such nucleic acids and/or vectors and plasmids. Theprovided methods include, for example, methods of making, identifying,or isolating molecules that bind to alpha4beta7, such asanti-alpha4beta7 antibodies, methods of determining whether a moleculebinds to alpha4beta7, methods of determining whether a molecule agonizesor antagonizes alpha4beta7, methods of making compositions, such aspharmaceutical compositions, comprising a molecule that binds toalpha4beta7, and methods for administering a molecule that bindsalpha4beta7 to a subject, for example, methods for treating a conditionmediated by alpha4beta7, and for agonizing or antagonizing a biologicalactivity of alpha4beta7, in vivo or in vitro.

Polynucleotide and polypeptide sequences are indicated using standardone- or three-letter abbreviations. Unless otherwise indicated, eachpolypeptide sequence has amino terminus at the left and a carboxyterminus at the right; each single-stranded nucleic acid sequence, andthe top strand of each double-stranded nucleic acid sequence has a 5′terminus at the left and a 3′ terminus at the right. A particularpolypeptide or polynucleotide sequence also can be described byexplaining how it differs from a reference sequence.

Unless otherwise defined herein, scientific and technical terms used inconnection with the present invention shall have the meanings that arecommonly understood by those of ordinary skill in the art. Further,unless otherwise required by context, singular terms shall includepluralities and plural terms shall include the singular. Generally,nomenclatures used in connection with, and techniques of, cell andtissue culture, molecular biology, immunology, microbiology, geneticsand protein and nucleic acid chemistry and hybridization describedherein are those well known and commonly used in the art. The methodsand techniques of the present invention are generally performedaccording to conventional methods well known in the art and as describedin various general and more specific references that are cited anddiscussed throughout the present specification unless otherwiseindicated. See, e.g., Sambrook et al. Molecular Cloning: A LaboratoryManual, 2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y. (1989) and Ausubel et al., Current Protocols in Molecular Biology,Greene Publishing Associates (1992), and Harlow and Lane Antibodies: ALaboratory Manual Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. (1990), which are incorporated herein by reference.Enzymatic reactions and purification techniques are performed accordingto manufacturer's specifications, as commonly accomplished in the art oras described herein. The terminology used in connection with, and thelaboratory procedures and techniques of, analytical chemistry, syntheticorganic chemistry, and medicinal and pharmaceutical chemistry describedherein are those well known and commonly used in the art. Standardtechniques can be used for chemical syntheses, chemical analyses,pharmaceutical preparation, formulation, and delivery, and treatment ofpatients.

The following terms, unless otherwise indicated, shall be understood tohave the following meanings:

The term “isolated molecule” (where the molecule is, for example, apolypeptide, a polynucleotide, or an antibody) is a molecule that byvirtue of its origin or source of derivation (1) is not associated withnaturally associated components that accompany it in its native state,(2) is substantially free of other molecules from the same species (3)is expressed by a cell from a different species, or (4) does not occurin nature without human intervention. Thus, a molecule that ischemically synthesized, or synthesized in a cellular system differentfrom the cell from which it naturally originates, will be “isolated”from its naturally associated components. A molecule also may berendered substantially free of naturally associated components byisolation, using purification techniques well known in the art. Moleculepurity or homogeneity may be assayed by a number of means well known inthe art. For example, the purity of a polypeptide sample may be assayedusing polyacrylamide gel electrophoresis and staining of the gel tovisualize the polypeptide using techniques well known in the art. Forcertain purposes, higher resolution may be provided by using HPLC orother means well known in the art for purification.

The terms “alpha4beta7 inhibitor” and “alpha4beta7 antagonist” are usedinterchangeably. Each is a molecule that detectably inhibits at leastone function of alpha4beta7. Conversely, an “alpha4beta7 agonist” is amolecule that detectably increases at least one function of alpha4beta7.The inhibition caused by an alpha4beta7 inhibitor need not be completeso long as it is detectable, for example by using an assay. Any assay ofa function of alpha4beta7 can be used, examples of which are providedherein. Examples of functions of alpha4beta7 that can be inhibited by analpha4beta7 inhibitor (or increased by an alpha4beta7 agonist) includeligand binding (i.e., binding to MAdCAM-1), adhesion toligand-expressing cells, trafficking to a particular compartment such asthe gut, release of cytokines, chemokines and other mediators, enhancingor exacerbating inflammatory response and tissue damage, and so on.Examples of types of alpha4beta7 inhibitors and alpha4beta7 agonistsinclude, but are not limited to, alpha4beta7 binding polypeptides suchas antigen binding proteins (e.g., alpha4beta7 antigen bindingproteins), antibodies, antibody fragments, and antibody derivatives.

The terms “peptide,” “polypeptide” and “protein” each refers to amolecule comprising two or more amino acid residues joined to each otherby peptide bonds. These terms encompass, e.g., native and artificialproteins, protein fragments and polypeptide analogs (such as muteins,variants, and fusion proteins) of a protein sequence as well aspost-translationally, or otherwise covalently or noncovalently, modifiedproteins. A peptide, polypeptide, or protein may be monomeric orpolymeric.

The term “polypeptide fragment” as used herein refers to a polypeptidethat has an amino-terminal and/or carboxy-terminal deletion as comparedto a corresponding full-length protein. Fragments can be, for example,at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 50, 70, 80, 90, 100,150 or 200 amino acids in length. Fragments can also be, for example, atmost 1,000, 750, 500, 250, 200, 175, 150, 125, 100, 90, 80, 70, 60, 50,40, 30, 20, 15, 14, 13, 12, 11, or 10 amino acids in length. Fragmentscan also result from proteolytic (or other) processing, which, forexample, results in variation in the amino and/or carboxy terminus offrom one to five amino acids from that predicted. A fragment can furthercomprise, at either or both of its ends, one or more additional aminoacids, for example, a sequence of amino acids from a differentnaturally-occurring protein (e.g., an Fc or leucine zipper domain) or anartificial amino acid sequence (e.g., an artificial linker sequence or atag protein).

Polypeptides of the invention include polypeptides that have beenmodified in any way and for any reason, for example, to: (1) reducesusceptibility to proteolysis, (2) reduce susceptibility to oxidation,(3) alter binding affinity for forming protein complexes, (4) alterbinding affinities, and (4) confer or modify other physicochemical orfunctional properties. Analogs include muteins of a polypeptide. Forexample, single or multiple amino acid substitutions (e.g., conservativeamino acid substitutions) may be made in the naturally occurringsequence (e.g., in the portion of the polypeptide outside the domain(s)forming intermolecular contacts). Consensus sequences can be used toselect amino acid residues for substitution; those of skill in the artrecognize that additional amino acid residues may also be substituted.

A “conservative amino acid substitution” is one that does notsubstantially change the structural characteristics of the parentsequence (e.g., a replacement amino acid should not tend to break ahelix that occurs in the parent sequence, or disrupt other types ofsecondary structure that characterize the parent sequence or arenecessary for its functionality). Examples of art-recognized polypeptidesecondary and tertiary structures are described in Proteins, Structuresand Molecular Principles (Creighton, Ed., W.H. Freeman and Company, NewYork (1984)); Introduction to Protein Structure (C. Branden and J.Tooze, eds., Garland Publishing, New York, N.Y. (1991)); and Thornton etal. Nature 354:105 (1991), which are each incorporated herein byreference.

The present invention also provides non-peptide analogs of alpha4beta7binding polypeptides. Non-peptide analogs are commonly used in thepharmaceutical industry as drugs with properties analogous to those ofthe template peptide. These types of non-peptide compound are termed“peptide mimetics” or “peptidomimetics,” see, for example, Fauchere, J.Adv. Drug Res. 15:29 (1986); Veber and Freidinger TINS p. 392 (1985);and Evans et al. J. Med. Chem. 30:1229 (1987), which are incorporatedherein by reference. Peptide mimetics that are structurally similar totherapeutically useful peptides may be used to produce an equivalenttherapeutic or prophylactic effect. Generally, peptidomimetics arestructurally similar to a paradigm polypeptide (i.e., a polypeptide thathas a desired biochemical property or pharmacological activity), such asa human antibody, but have one or more peptide linkages optionallyreplaced by a linkage selected from the group consisting of: —CH₂NH—,—CH₂S—, —CH₂—CH₂—, —CH═CH-(cis and trans), —COCH₂—, —CH(OH)CH₂—, and—CH₂SO—, by methods well known in the art. Systematic substitution ofone or more amino acids of a consensus sequence with a D-amino acid ofthe same type (e.g., D-lysine in place of L-lysine) may also be used togenerate more stable peptides. In addition, constrained peptidescomprising a consensus sequence or a substantially identical consensussequence variation may be generated by methods known in the art (Rizoand Gierasch Ann. Rev. Biochem. 61:387 (1992), incorporated herein byreference), for example, by adding internal cysteine residues capable offorming intramolecular disulfide bridges which cyclize the peptide.

A “variant” of a polypeptide (e.g., an antibody) comprises an amino acidsequence wherein one or more amino acid residues are inserted into,deleted from and/or substituted into the amino acid sequence relative toanother polypeptide sequence. Variants of the invention include fusionproteins.

A “derivative” of a polypeptide is a polypeptide (e.g., an antibody)that has been chemically modified, e.g., via conjugation to anotherchemical moiety (such as, for example, polyethylene glycol or albumin,e.g., human serum albumin), phosphorylation, and/or glycosylation.Unless otherwise indicated, the term “antibody” includes, in addition toantibodies comprising two full-length heavy chains and two full-lengthlight chains, derivatives, variants, fragments, and muteins thereof,examples of which are described below.

An “antigen binding protein” is a protein comprising a portion thatbinds to an antigen and, optionally, a scaffold or framework portionthat allows the antigen binding portion to adopt a conformation thatpromotes binding of the antigen binding protein to the antigen. Examplesof antigen binding proteins include antibodies, antibody fragments(e.g., an antigen binding portion of an antibody), antibody derivatives,and antibody analogs. The antigen binding protein can comprise, forexample, an alternative protein scaffold or artificial scaffold withgrafted CDRs or CDR derivatives. Such scaffolds include, but are notlimited to, antibody-derived scaffolds comprising mutations introducedto, for example, stabilize the three-dimensional structure of theantigen binding protein as well as wholly synthetic scaffoldscomprising, for example, a biocompatible polymer. See, for example,Korndorfer et al., 2003, Proteins: Structure, Function, andBioinformatics, Volume 53, Issue 1:121-129; Roque et al., 2004,Biotechnol. Prog. 20:639-654. In addition, peptide antibody mimetics(“PAMs”) can be used, as well as scaffolds based on antibody mimeticsutilizing fibronection components as a scaffold.

An antigen binding protein can have, for example, the structure of anaturally occurring immunoglobulin. An “immunoglobulin” is a tetramericmolecule. In a naturally occurring immunoglobulin, each tetramer iscomposed of two identical pairs of polypeptide chains, each pair havingone “light” (about 25 kDa) and one “heavy” chain (about 50-70 kDa). Theamino-terminal portion of each chain includes a variable region of about100 to 110 or more amino acids primarily responsible for antigenrecognition. The carboxy-terminal portion of each chain defines aconstant region primarily responsible for effector function. Human lightchains are classified as kappa or lambda light chains. Heavy chains areclassified as mu, delta, gamma, alpha, or epsilon, and define theantibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. Withinlight and heavy chains, the variable and constant regions are joined bya “J” region of about 12 or more amino acids, with the heavy chain alsoincluding a “D” region of about 10 more amino acids. See generally,Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y.(1989)) (incorporated by reference in its entirety for all purposes).The variable regions of each light/heavy chain pair form the antibodybinding site such that an intact immunoglobulin has two binding sites.

The variable regions of naturally occurring immunoglobulin chainsexhibit the same general structure of relatively conserved frameworkregions (FR) joined by three hypervariable regions, also calledcomplementarity determining regions or CDRs. From N-terminus toC-terminus, both light and heavy chains comprise the domains FR1, CDR1,FR2, CDR2, FR3, CDR3 and FR4. The assignment of amino acids to eachdomain is in accordance with the definitions of Kabat et al. inSequences of Proteins of Immunological Interest, 5^(th) Ed., US Dept. ofHealth and Human Services, PHS, NIH, NIH Publication no. 91-3242, 1991.Other numbering systems for the amino acids in immunoglobulin chainsinclude IMGT® (the international ImMunoGeneTics information system;Lefranc et al, Dev. Comp. Immunol. 29:185-203; 2005) and AHo (Honeggerand Pluckthun, J. Mol. Biol. 309(3):657-670; 2001).

Antibodies can be obtained from sources such as serum or plasma thatcontain immunoglobulins having varied antigenic specificity. If suchantibodies are subjected to affinity purification, they can be enrichedfor a particular antigenic specificity. Such enriched preparations ofantibodies usually are made of less than about 10% antibody havingspecific binding activity for the particular antigen. Subjecting thesepreparations to several rounds of affinity purification can increase theproportion of antibody having specific binding activity for the antigen.Antibodies prepared in this manner are often referred to as“monospecific.” Monospecific antibody preparations can be made up ofabout 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%,99%, or 99.9% antibody having specific binding activity for theparticular antigen.

An “antibody” refers to an intact immunoglobulin or to an antigenbinding portion thereof that competes with the intact antibody forspecific binding, unless otherwise specified. Antigen binding portionsmay be produced by recombinant DNA techniques or by enzymatic orchemical cleavage of intact antibodies. Antigen binding portionsinclude, inter alia, Fab, Fab′, F(ab′)₂, Fv, domain antibodies (dAbs),and complementarity determining region (CDR) fragments, variable regionfragments, single-chain antibodies (scFv), chimeric antibodies,diabodies, triabodies, tetrabodies, and polypeptides that contain atleast a portion of an immunoglobulin that is sufficient to conferspecific antigen binding to the polypeptide.

A Fab fragment is a monovalent fragment having the V_(L), V_(H), C_(L)and C_(H)1 domains; a F(ab′)₂ fragment is a bivalent fragment having twoFab fragments linked by a disulfide bridge at the hinge region; a Fdfragment has the V_(H) and C_(H)1 domains; an Fv fragment has the V_(L)and V_(H) domains of a single arm of an antibody; and a dAb fragment hasa V_(H) domain, a V_(L) domain, or an antigen-binding fragment of aV_(H) or V_(L) domain (U.S. Pat. Nos. 6,846,634, 6,696,245, US App. Pub.No. 05/0202512, 04/0202995, 04/0038291, 04/0009507, 03/0039958, Ward etal., Nature 341:544-546, 1989).

A single-chain antibody (scFv) is an antibody in which a V_(L) and aV_(H) region are joined via a linker (e.g., a synthetic sequence ofamino acid residues) to form a continuous protein chain wherein thelinker is long enough to allow the protein chain to fold back on itselfand form a monovalent antigen binding site (see, e.g., Bird et al.,1988, Science 242:423-26 and Huston et al., 1988, Proc. Natl. Acad. Sci.USA 85:5879-83). Diabodies are bivalent antibodies comprising twopolypeptide chains, wherein each polypeptide chain comprises V_(H) andV_(L) domains joined by a linker that is too short to allow for pairingbetween two domains on the same chain, thus allowing each domain to pairwith a complementary domain on another polypeptide chain (see, e.g.,Holliger et al., 1993, Proc. Natl. Acad. Sci. USA 90:6444-48, and Poljaket al., 1994, Structure 2:1121-23). If the two polypeptide chains of adiabody are identical, then a diabody resulting from their pairing willhave two identical antigen binding sites. Polypeptide chains havingdifferent sequences can be used to make a diabody with two differentantigen binding sites. Similarly, triabodies and tetrabodies areantibodies comprising three and four polypeptide chains, respectively,and forming three and four antigen binding sites, respectively, whichcan be the same or different.

Complementarity determining regions (CDRs) and framework regions (FR) ofa given antibody may be identified using the system described by Kabatet al. supra; Lefranc et al., supra and/or Honegger and Pluckthun,supra. One or more CDRs may be incorporated into a molecule eithercovalently or noncovalently to make it an antigen binding protein. Anantigen binding protein may incorporate the CDR(s) as part of a largerpolypeptide chain, may covalently link the CDR(s) to another polypeptidechain, or may incorporate the CDR(s) noncovalently. The CDRs permit theantigen binding protein to specifically bind to a particular antigen ofinterest.

An antigen binding protein may have one or more binding sites. If thereis more than one binding site, the binding sites may be identical to oneanother or may be different. For example, a naturally occurring humanimmunoglobulin typically has two identical binding sites, while a“bispecific” or “bifunctional” antibody has two different binding sites.

The term “human antibody” includes all antibodies that have one or morevariable and constant regions derived from human immunoglobulinsequences. In one embodiment, all of the variable and constant domainsare derived from human immunoglobulin sequences (a fully humanantibody). These antibodies may be prepared in a variety of ways,examples of which are described below, including through theimmunization with an antigen of interest of a mouse that is geneticallymodified to express antibodies derived from human heavy and/or lightchain-encoding genes.

A humanized antibody has a sequence that differs from the sequence of anantibody derived from a non-human species by one or more amino acidsubstitutions, deletions, and/or additions, such that the humanizedantibody is less likely to induce an immune response, and/or induces aless severe immune response, as compared to the non-human speciesantibody, when it is administered to a human subject. In one embodiment,certain amino acids in the framework and constant domains of the heavyand/or light chains of the non-human species antibody are mutated toproduce the humanized antibody. In another embodiment, the constantdomain(s) from a human antibody are fused to the variable domain(s) of anon-human species. In another embodiment, one or more amino acidresidues in one or more CDR sequences of a non-human antibody arechanged to reduce the likely immunogenicity of the non-human antibodywhen it is administered to a human subject, wherein the changed aminoacid residues either are not critical for immunospecific binding of theantibody to its antigen, or the changes to the amino acid sequence thatare made are conservative changes, such that the binding of thehumanized antibody to the antigen is not significantly worse than thebinding of the non-human antibody to the antigen. Examples of how tomake humanized antibodies may be found in U.S. Pat. Nos. 6,054,297,5,886,152 and 5,877,293.

The term “chimeric antibody” refers to an antibody that contains one ormore regions from one antibody and one or more regions from one or moreother antibodies. In one embodiment, one or more of the CDRs are derivedfrom a human anti-alpha4beta7 antibody. In another embodiment, all ofthe CDRs are derived from a human anti-alpha4beta7 antibody. In anotherembodiment, the CDRs from more than one human anti-alpha4beta7antibodies are mixed and matched in a chimeric antibody. For instance, achimeric antibody may comprise a CDR1 from the light chain of a firsthuman anti-alpha4beta7 antibody, a CDR2 and a CDR3 from the light chainof a second human anti-alpha4beta7 antibody, and the CDRs from the heavychain from a third anti-alpha4beta7 antibody. Other combinations arepossible and are included within the embodiments of the invention.

Further, the framework regions may be derived from one of the sameanti-alpha4beta7 antibodies, from one or more different antibodies, suchas a human antibody, or from a humanized antibody. In one example of achimeric antibody, a portion of the heavy and/or light chain isidentical with, homologous to, or derived from an antibody from aparticular species or belonging to a particular antibody class orsubclass, while the remainder of the chain(s) is/are identical with,homologous to, or derived from an antibody (-ies) from another speciesor belonging to another antibody class or subclass. Also included arefragments of such antibodies that exhibit the desired biologicalactivity (i.e., the ability to specifically bind alpha4beta7). See,e.g., U.S. Pat. No. 4,816,567 and Morrison, 1985, Science 229:1202-07.

A “neutralizing antibody” or an “inhibitory antibody” is an antibodythat inhibits the interaction of alpha4beta7 with MAdCAM-1 when anexcess of the anti-alpha4beta7 antibody reduces the amount ofinteraction by at least about 20% using an assay such as those describedherein in the Examples. In various embodiments, the antigen bindingprotein reduces the interaction of alpha4beta7 with MAdCAM-1 alpha4beta7by at least 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%,and 99.9%.

Fragments or analogs of antibodies can be readily prepared by those ofordinary skill in the art following the teachings of this specificationand using techniques well-known in the art Amino- and carboxy-termini offragments or analogs occur near boundaries of functional domains.Structural and functional domains can be identified by comparison of thenucleotide and/or amino acid sequence data to public or proprietarysequence databases. Computerized comparison methods can be used toidentify sequence motifs or predicted protein conformation domains thatoccur in other proteins of known structure and/or function. Methods toidentify protein sequences that fold into a known three-dimensionalstructure are known. See, e.g., Bowie et al., 1991, Science 253:164.

A “CDR grafted antibody” is an antibody comprising one or more CDRsderived from an antibody of a particular species or isotype and theframework of another antibody of the same or different species orisotype.

A “multi-specific antibody” is an antibody that recognizes more than oneepitope on one or more antigens. A subclass of this type of antibody isa “bi-specific antibody” which recognizes two distinct epitopes on thesame or different antigens.

An antigen binding protein “specifically binds” to an antigen (e.g.,human alpha4beta7) if it binds to the antigen with a dissociationconstant of 1 nanomolar or less. As used herein, an antigen bindingprotein is “heterodimer specific” if it binds to a first heterodimericintegrin but not to other integrins that share one chain with the firstintegrin. For example, an antibody that is alpha4beta7 heterodimerspecific will bind to alpha4beta7 but not to alpha4beta1 or alphaEbeta7.

Integrins are known to adapt different conformations, depending on theactivation state of the cell(s) expressing them and on the presence orabsence of certain metal ions. An integrin in “active” conformationbinds to its cognate ligand with higher affinity than the same integrinin “inactive” conformation. An antigen binding protein may bind to anintegrin in only its active conformation, in only its inactiveconformation, or in both or either conformations. For example, analpha4beta7 heterodimer specific antigen binding protein may bindalpha4beta7 in the presence or absence of the divalent cationmanganese²⁺ (Mn²⁺), indicating that the antigen binding protein bindsboth active and inactive alpah4beta7.

An “antigen binding domain,” “antigen binding region,” or “antigenbinding site” is a portion of an antigen binding protein that containsamino acid residues (or other moieties) that interact with an antigenand contribute to the antigen binding protein's specificity and affinityfor the antigen. For an antibody that specifically binds to its antigen,this will include at least part of at least one of its CDR domains.

An “epitope” is the portion of a molecule that is bound by an antigenbinding protein (e.g., by an antibody). An epitope can comprisenon-contiguous portions of the molecule (e.g., in a polypeptide, aminoacid residues that are not contiguous in the polypeptide's primarysequence but that, in the context of the polypeptide's tertiary andquaternary structure, are near enough to each other to be bound by anantigen binding protein).

The “percent identity” of two polynucleotide or two polypeptidesequences is determined by comparing the sequences using the GAPcomputer program (a part of the GCG Wisconsin Package, version 10.3(Accelrys, San Diego, Calif.)) using its default parameters.

The terms “polynucleotide,” “oligonucleotide” and “nucleic acid” areused interchangeably throughout and include DNA molecules (e.g., cDNA orgenomic DNA), RNA molecules (e.g., mRNA), analogs of the DNA or RNAgenerated using nucleotide analogs (e.g., peptide nucleic acids andnon-naturally occurring nucleotide analogs), and hybrids thereof. Thenucleic acid molecule can be single-stranded or double-stranded. In oneembodiment, the nucleic acid molecules of the invention comprise acontiguous open reading frame encoding an antibody, or a fragment,derivative, mutein, or variant thereof, of the invention.

Two single-stranded polynucleotides are “the complement” of each otherif their sequences can be aligned in an anti-parallel orientation suchthat every nucleotide in one polynucleotide is opposite itscomplementary nucleotide in the other polynucleotide, without theintroduction of gaps, and without unpaired nucleotides at the 5′ or the3′ end of either sequence. A polynucleotide is “complementary” toanother polynucleotide if the two polynucleotides can hybridize to oneanother under moderately stringent conditions. Thus, a polynucleotidecan be complementary to another polynucleotide without being itscomplement.

A “vector” is a nucleic acid that can be used to introduce anothernucleic acid linked to it into a cell. One type of vector is a“plasmid,” which refers to a linear or circular double stranded DNAmolecule into which additional nucleic acid segments can be ligated.Another type of vector is a viral vector (e.g., replication defectiveretroviruses, adenoviruses and adeno-associated viruses), whereinadditional DNA segments can be introduced into the viral genome. Certainvectors are capable of autonomous replication in a host cell into whichthey are introduced (e.g., bacterial vectors comprising a bacterialorigin of replication and episomal mammalian vectors). Other vectors(e.g., non-episomal mammalian vectors) are integrated into the genome ofa host cell upon introduction into the host cell, and thereby arereplicated along with the host genome. An “expression vector” is a typeof vector that can direct the expression of a chosen polynucleotide.

A nucleotide sequence is “operably linked” to a regulatory sequence ifthe regulatory sequence affects the expression (e.g., the level, timing,or location of expression) of the nucleotide sequence. A “regulatorysequence” is a nucleic acid that affects the expression (e.g., thelevel, timing, or location of expression) of a nucleic acid to which itis operably linked. The regulatory sequence can, for example, exert itseffects directly on the regulated nucleic acid, or through the action ofone or more other molecules (e.g., polypeptides that bind to theregulatory sequence and/or the nucleic acid). Examples of regulatorysequences include promoters, enhancers and other expression controlelements (e.g., polyadenylation signals). Further examples of regulatorysequences are described in, for example, Goeddel, 1990, Gene ExpressionTechnology: Methods in Enzymology 185, Academic Press, San Diego, Calif.and Baron et al., 1995, Nucleic Acids Res. 23:3605-06.

A “host cell” is a cell that can be used to express a nucleic acid,e.g., a nucleic acid of the invention. A host cell can be a prokaryote,for example, E. coli, or it can be a eukaryote, for example, asingle-celled eukaryote (e.g., a yeast or other fungus), a plant cell(e.g., a tobacco or tomato plant cell), an animal cell (e.g., a humancell, a monkey cell, a hamster cell, a rat cell, a mouse cell, or aninsect cell) or a hybridoma. Examples of host cells include the COS-7line of monkey kidney cells (ATCC CRL 1651) (see Gluzman et al., 1981,Cell 23:175), L cells, C127 cells, 3T3 cells (ATCC CCL 163), Chinesehamster ovary (CHO) cells or their derivatives such as Veggie CHO andrelated cell lines which grow in serum-free media (see Rasmussen et al.,1998, Cytotechnology 28:31) or CHO strain DX-B11, which is deficient inDHFR (see Urlaub et al., 1980, Proc. Natl. Acad. Sci. USA 77:4216-20),HeLa cells, BHK (ATCC CRL 10) cell lines, the CV1/EBNA cell line derivedfrom the African green monkey kidney cell line CV1 (ATCC CCL 70) (seeMcMahan et al., 1991, EMBO J. 10:2821), human embryonic kidney cellssuch as 293, 293 EBNA or MSR 293, human epidermal A431 cells, humanColo205 cells, other transformed primate cell lines, normal diploidcells, cell strains derived from in vitro culture of primary tissue,primary explants, HL-60, U937, HaK or Jurkat cells. Typically, a hostcell is a cultured cell that can be transformed or transfected with apolypeptide-encoding nucleic acid, which can then be expressed in thehost cell. The phrase “recombinant host cell” can be used to denote ahost cell that has been transformed or transfected with a nucleic acidto be expressed. A host cell also can be a cell that comprises thenucleic acid but does not express it at a desired level unless aregulatory sequence is introduced into the host cell such that itbecomes operably linked with the nucleic acid. It is understood that theterm host cell refers not only to the particular subject cell but alsoto the progeny or potential progeny of such a cell. Because certainmodifications may occur in succeeding generations due to, e.g., mutationor environmental influence, such progeny may not, in fact, be identicalto the parent cell, but are still included within the scope of the termas used herein.

Antigen Binding Proteins

In one aspect, the present invention provides antigen binding proteins(e.g., antibodies, antibody fragments, antibody derivatives, antibodymuteins, and antibody variants) that bind to alpha4beta7, e.g., humanalpha4beta7.

Antigen binding proteins in accordance with the present inventioninclude antigen binding proteins that inhibit a biological activity ofalpha4beta7. Examples of such biological activities include binding ofalpha4beta7 to MAdCAM-1, and adhesion between cells expressingalpha4beta7 and those expressing MAdCAM-1. Other biological activitiesinclude those mediated by alpha4beta7 in vivo, such as trafficking orhoming; in particular, alpha4beta7 is involved in the trafficking oflymphocytes to the gut, Increased MAdCAM-1 expression in the inflamedgut enhances recruitment of alpha4beta7 expressing lymphocytes to thegut, where aberrant lymphocyte activation augments inflammatory responseand tissue damage.

Different antigen binding proteins may bind to different domains orepitopes of alpha4beta7 or act by different mechanisms of action.Examples include but are not limited to antigen binding proteins thatinterfere with the ability of alpha4beta7 to bind MAdCAM-1 or thatinhibit cellular interactions such as adhesion between cells expressingalpha4beta7 and cells expressing MAdCAM-1. The site of action may be,for example, intracellular (e.g., by interfering with an intracellularsignaling cascade) or extracellular. An antigen binding protein need notcompletely inhibit alpha4beta7 induced activity to find use in thepresent invention; rather, antigen binding proteins that reduce aparticular activity of alpha4beta7 are contemplated for use as well.(Discussions herein of particular mechanisms of action foralpha4beta7-binding antigen binding proteins in treating particulardiseases are illustrative only, and the methods presented herein are notbound thereby.)

Other derivatives of anti-alpha4beta7 antibodies within the scope ofthis invention include covalent or aggregative conjugates ofanti-alpha4beta7 antibodies, or fragments thereof, with other proteinsor polypeptides, such as by expression of recombinant fusion proteinscomprising heterologous polypeptides fused to the N-terminus orC-terminus of an anti-alpha4beta7 antibody polypeptide. For example, theconjugated peptide may be a heterologous signal (or leader) polypeptide,e.g., the yeast alpha-factor leader, or a peptide such as an epitopetag. Antigen binding protein-containing fusion proteins can comprisepeptides added to facilitate purification or identification of antigenbinding protein (e.g., poly-His). An antigen binding protein also can belinked to the FLAG® peptide Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys (DYKDDDDK)(SEQ ID NO:62) as described in Hopp et al., Bio/Technology 6:1204, 1988,and U.S. Pat. No. 5,011,912. The FLAG® peptide is highly antigenic andprovides an epitope reversibly bound by a specific monoclonal antibody(mAb), enabling rapid assay and facile purification of expressedrecombinant protein. Reagents useful for preparing fusion proteins inwhich the FLAG® peptide is fused to a given polypeptide are commerciallyavailable (Sigma-Aldrich, St. Louis Mo.).

Oligomers that contain one or more antigen binding proteins may beemployed as alpha4beta7 antagonists. Oligomers may be in the form ofcovalently-linked or non-covalently-linked dimers, trimers, or higheroligomers. Oligomers comprising two or more antigen binding protein arecontemplated for use, with one example being a homodimer. Otheroligomers include heterodimers, homotrimers, heterotrimers,homotetramers, heterotetramers, etc.

One embodiment is directed to oligomers comprising multiple antigenbinding proteins joined via covalent or non-covalent interactionsbetween peptide moieties fused to the antigen binding proteins. Suchpeptides may be peptide linkers (spacers), or peptides that have theproperty of promoting oligomerization. Leucine zippers and certainpolypeptides derived from antibodies are among the peptides that canpromote oligomerization of antigen binding proteins attached thereto, asdescribed in more detail below.

In particular embodiments, the oligomers comprise from two to fourantigen binding proteins. The antigen binding proteins of the oligomermay be in any form, such as any of the forms described above, e.g.,variants or fragments. Preferably, the oligomers comprise antigenbinding proteins that have alpha4beta7 binding activity.

In one embodiment, an oligomer is prepared using polypeptides derivedfrom immunoglobulins. Preparation of fusion proteins comprising certainheterologous polypeptides fused to various portions of antibody-derivedpolypeptides (including the Fc domain) has been described, e.g., byAshkenazi et al., 1991, PNAS USA 88:10535; Byrn et al., 1990, Nature344:677; and Hollenbaugh et al., 1992 “Construction of ImmunoglobulinFusion Proteins”, in Current Protocols in Immunology, Suppl. 4, pages10.19.1-10.19.11.

One embodiment of the present invention is directed to a dimercomprising two fusion proteins created by fusing an alpha4beta7 bindingfragment of an anti-alpha4beta7 antibody to the Fc region of anantibody. The dimer can be made by, for example, inserting a gene fusionencoding the fusion protein into an appropriate expression vector,expressing the gene fusion in host cells transformed with therecombinant expression vector, and allowing the expressed fusion proteinto assemble much like antibody molecules, whereupon interchain disulfidebonds form between the Fc moieties to yield the dimer.

The term “Fc polypeptide” as used herein includes native and muteinforms of polypeptides derived from the Fc region of an antibody.Truncated forms of such polypeptides containing the hinge region thatpromotes dimerization also are included. Fusion proteins comprising Fcmoieties (and oligomers formed therefrom) offer the advantage of facilepurification by affinity chromatography over Protein A or Protein Gcolumns.

One suitable Fc polypeptide, described in PCT application WO 93/10151(hereby incorporated by reference), is a single chain polypeptideextending from the N-terminal hinge region to the native C-terminus ofthe Fc region of a human IgG1 antibody. Another useful Fc polypeptide isthe Fc mutein described in U.S. Pat. No. 5,457,035 and in Baum et al.,1994, EMBO J. 13:3992-4001. The amino acid sequence of this mutein isidentical to that of the native Fc sequence presented in WO 93/10151,except that amino acid 19 has been changed from Leu to Ala, amino acid20 has been changed from Leu to Glu, and amino acid 22 has been changedfrom Gly to Ala. The mutein exhibits reduced affinity for Fc receptors.

In other embodiments, the variable portion of the heavy and/or lightchains of an anti-alpha4beta7 antibody may be substituted for thevariable portion of an antibody heavy and/or light chain.

Alternatively, the oligomer is a fusion protein comprising multipleantigen binding proteins, with or without peptide linkers (spacerpeptides). Among the suitable peptide linkers are those described inU.S. Pat. Nos. 4,751,180 and 4,935,233.

Another method for preparing oligomeric antigen binding proteinsinvolves use of a leucine zipper. Leucine zipper domains are peptidesthat promote oligomerization of the proteins in which they are found.Leucine zippers were originally identified in several DNA-bindingproteins (Landschulz et al., 1988, Science 240:1759), and have sincebeen found in a variety of different proteins. Among the known leucinezippers are naturally occurring peptides and derivatives thereof thatdimerize or trimerize. Examples of leucine zipper domains suitable forproducing soluble oligomeric proteins are described in PCT applicationWO 94/10308, and the leucine zipper derived from lung surfactant proteinD (SPD) described in Hoppe et al., 1994, FEBS Letters 344:191, herebyincorporated by reference. The use of a modified leucine zipper thatallows for stable trimerization of a heterologous protein fused theretois described in Fanslow et al., 1994, Semin. Immunol. 6:267-78. In oneapproach, recombinant fusion proteins comprising an anti-alpha4beta7antibody fragment or derivative fused to a leucine zipper peptide areexpressed in suitable host cells, and the soluble oligomericanti-alpha4beta7 antibody fragments or derivatives that form arerecovered from the culture supernatant.

In one aspect, the present invention provides antigen binding proteinsthat interfere with the binding of alpha4beta7 to MAdCAM-1. Such antigenbinding proteins can be made against alpha4beta7, or a fragment, variantor derivative thereof, and screened in conventional assays for theability to interfere with the binding of alpha4beta7 to MAdCAM-1.Examples of suitable assays are assays that test the antigen bindingproteins for the ability to inhibit binding of MAdCAM-1 (i.e., solubleMAdCAM-1) to cells expressing alpha4beta7, or that test antigen bindingproteins for the ability to reduce a biological or cellular responsethat results from the interaction of MAdCAM-1 and alpha4beta7 (i.e.,adhesion of cells expressing alpha4beta7 to MAdCAM-1, orMAdCAM-1-expressing cells). Additional assays that test the antigenbinding proteins include those that qualitatively or quantitativelycompare the binding of an antigen binding protein to a alpha4beta7polypeptide to the binding of a known antigen binding protein to aalpha4beta7 polypeptide, several examples of which are disclosed herein.

In another aspect, the present invention provides an antigen bindingprotein that demonstrates species selectivity. In one embodiment, theantigen binding protein binds to one or more mammalian alpha4beta7, forexample, to human alpha4beta7 and one or more of mouse, rat, guinea pig,hamster, gerbil, cat, rabbit, dog, goat, sheep, cow, horse, camel, andnon-human primate alpha4beta7. In another embodiment, the antigenbinding protein binds to one or more primate alpha4beta7, for example,to human alpha4beta7 and one or more of cynomologous, marmoset, rhesus,tamarin and chimpanzee alpha4beta7. In another embodiment, the antigenbinding protein binds specifically to human, cynomologous, marmoset,rhesus, tamarin or chimpanzee alpha4beta7. In another embodiment, theantigen binding protein does not bind to one or more of mouse, rat,guinea pig, hamster, gerbil, cat, rabbit, dog, goat, sheep, cow, horse,camel, and non-human primate alpha4beta7. In another embodiment, theantigen binding protein does not bind to a New World monkey species suchas a marmoset.

In another embodiment, the antigen binding protein does not exhibitspecific binding to any naturally occurring protein other thanalpha4beta7. In another embodiment, the antigen binding protein does notexhibit specific binding to any naturally occurring protein other thanmammalian alpha4beta7. In another embodiment, the antigen bindingprotein does not exhibit specific binding to any naturally occurringprotein other than primate alpha4beta7. In another embodiment, theantigen binding protein does not exhibit specific binding to anynaturally occurring protein other than human alpha4beta7. In anotherembodiment, the antigen binding protein specifically binds toalpha4beta7 from at least one non-human primate, for example,cynomologous monkey, and human alpha4beta7. In another embodiment, theantigen binding protein specifically binds to non-human primate,cynomologous monkey, and human alpha4beta7 with a similar bindingaffinity. In another embodiment, the antigen binding protein blocks anactivity of non-human primate, cynomologous monkey, and humanalpha4beta7. In another embodiment, the antigen binding protein has asimilar IC₅₀ or EC₅₀ against non-human primate, cynomologous monkey, andhuman alpha4beta7 in an assay as described herein.

One may determine the selectivity of an antigen binding protein for analpha4beta7 using methods well known in the art and following theteachings of the specification. For example, one may determine theselectivity using Western blot, FACS, ELISA or RIA.

In another aspect, the present invention provides an alpha4beta7 bindingantigen binding protein (for example, an anti-alpha4beta7 antibody),that has one or more of the following characteristics: binds to bothhuman and non-human primate alpha4beta7, inhibits binding of MAdCAM-1 toalpha4beta7, inhibits the adhesion of cells expressing alpha4beta7 toMAdCAM-1, inhibits the adhesion of cells expressing alpha4beta7 to cellsexpressing MAdCAM-1, inhibits trafficking of cells expressingalpha4beta7 to tissues comprising cells expressing MAdCAM-1, binds bothactive and inactive forms of alpha4beta7, causes relatively littledown-regulation of cell-surface expressed alpha4beta7.

Antigen-binding fragments of antigen binding proteins of the inventionmay be produced by conventional techniques. Examples of such fragmentsinclude, but are not limited to, Fab and F(ab′)₂ fragments. Antibodyfragments and derivatives produced by genetic engineering techniquesalso are contemplated.

Additional embodiments include chimeric antibodies, e.g., humanizedversions of non-human (e.g., murine) monoclonal antibodies. Suchhumanized antibodies may be prepared by known techniques, and offer theadvantage of reduced immunogenicity when the antibodies are administeredto humans. In one embodiment, a humanized monoclonal antibody comprisesthe variable domain of a murine antibody (or all or part of the antigenbinding site thereof) and a constant domain derived from a humanantibody. Alternatively, a humanized antibody fragment may comprise theantigen binding site of a murine monoclonal antibody and a variabledomain fragment (lacking the antigen-binding site) derived from a humanantibody. Procedures for the production of chimeric and furtherengineered monoclonal antibodies include those described in Riechmann etal., 1988, Nature 332:323, Liu et al., 1987, Proc. Nat. Acad. Sci. USA84:3439, Larrick et al., 1989, Bio/Technology 7:934, and Winter et al.,1993, TIPS 14:139. In one embodiment, the chimeric antibody is a CDRgrafted antibody. Techniques for humanizing antibodies are discussed in,e.g., U.S. patent application Ser. No. 10/194,975 (published Feb. 27,2003), U.S. Pat. Nos. 5,869,619, 5,225,539, 5,821,337, 5,859,205, Padlanet al., 1995, FASEB J. 9:133-39, and Tamura et al., 2000, J. Immunol.164:1432-41.

Procedures have been developed for generating human or partially humanantibodies in non-human animals. For example, mice in which one or moreendogenous immunoglobulin genes have been inactivated by various meanshave been prepared. Human immunoglobulin genes have been introduced intothe mice to replace the inactivated mouse genes. Antibodies produced inthe animal incorporate human immunoglobulin polypeptide chains encodedby the human genetic material introduced into the animal. In oneembodiment, a non-human animal, such as a transgenic mouse, is immunizedwith an alpha4beta7 polypeptide, such that antibodies directed againstthe alpha4beta7 polypeptide are generated in the animal. One example ofa suitable immunogen is a soluble human alpha4beta7, such as apolypeptide comprising a portion of alpha4beta7, or other immunogenicfragment alpha4beta7. Another example of a suitable immunogen is cellsexpressing high levels of alpha4beta7, or cell membrane preparationstherefrom.

Examples of techniques for production and use of transgenic animals forthe production of human or partially human antibodies are described inU.S. Pat. Nos. 5,814,318, 5,569,825, and 5,545,806, Davis et al., 2003,Production of human antibodies from transgenic mice in Lo, ed. AntibodyEngineering Methods and Protocols, Humana Press, N.J.: 191-200,Kellermann et al., 2002, Curr Opin Biotechnol. 13:593-97, Russel et al.,2000, Infect Immun. 68:1820-26, Gallo et al., 2000, Eur J. Immun.30:534-40, Davis et al., 1999, Cancer Metastasis Rev. 18:421-25, Green,1999, J Immunol Methods. 231:11-23, Jakobovits, 1998, Adv Drug Deliv Rev31:33-42, Green et al., 1998, J Exp Med. 188:483-95, Jakobovits A, 1998,Exp. Opin. Invest. Drugs. 7:607-14, Tsuda et al., 1997, Genomics42:413-21, Mendez et al., 1997, Nat. Genet. 15:146-56, Jakobovits, 1994,Curr Biol. 4:761-63, Arbones et al., 1994, Immunity. 1:247-60, Green etal., 1994, Nat. Genet. 7:13-21, Jakobovits et al., 1993, Nature362:255-58, Jakobovits et al., 1993, Proc Natl Acad Sci USA. 90:2551-55.Chen, J. et al., 1993, Int Immunol 5: 647-656, Choi et al., 1993, NatureGenetics 4: 117-23, Fishwild et al., 1996, Nat Biotechnol 14: 845-51,Harding et al., 1995, Ann NY Acad Sci, Lonberg et al., 1994, Nature 368:856-59, Lonberg, 1994, Transgenic Approaches to Human MonoclonalAntibodies in Handbook of Experimental Pharmacology 113: 49-101, Lonberget al., 1995, Int Rev Immunol 13: 65-93, Neuberger, 1996, Nat Biotechnol14: 826, Taylor et al., 1992, Nucleic Acids Research 20: 6287-95, Tayloret al., 1994, Int Immunol 6: 579-91, Tomizuka et al., 1997, Nat Gen 16:133-43, Tomizuka et al., 2000, Proc Natl Acad Sci USA. 97: 722-27,Tuaillon et al., 1993, Proc Natl Acad Sci USA. 90: 3720-24, and Tuaillonet al., 1994, J Immunol 152: 2912-20. These and other examples are alsodiscussed in U.S. Patent application publication 2007-0098715, publishedMay 3, 2007.

In another aspect, the present invention provides monoclonal antibodiesthat bind to alpha4beta7. Monoclonal antibodies may be produced usingany technique known in the art, e.g., by immortalizing spleen cellsharvested from the transgenic animal after completion of theimmunization schedule. The spleen cells can be immortalized using anytechnique known in the art, e.g., by fusing them with myeloma cells toproduce hybridomas. Myeloma cells for use in hybridoma-producing fusionprocedures preferably are non-antibody-producing, have high fusionefficiency, and enzyme deficiencies that render them incapable ofgrowing in certain selective media which support the growth of only thedesired fused cells (hybridomas). Examples of suitable cell lines foruse in mouse fusions include Sp-20, P3-X63/Ag8, P3-X63-Ag8.653, NS1/1.Ag4 1, Sp210-Ag14, FO, NSO/U, MPC-11, MPC11-X45-GTG 1.7 and S194/5XX0 Bul;examples of cell lines used in rat fusions include R210.RCY3, Y3-Ag1.2.3, IR983F and 4B210. Other cell lines useful for cell fusions areU-266, GM1500-GRG2, LICR-LON-HMy2 and UC729-6.

In one embodiment, a hybridoma cell line is produced by immunizing ananimal (e.g., a transgenic animal having human immunoglobulin sequences)with an alpha4beta7 immunogen; harvesting spleen cells from theimmunized animal; fusing the harvested spleen cells to a myeloma cellline, thereby generating hybridoma cells; establishing hybridoma celllines from the hybridoma cells, and identifying a hybridoma cell linethat produces an antibody that binds an alpha4beta7 polypeptide. Suchhybridoma cell lines, and anti-alpha4beta7 monoclonal antibodiesproduced by them, are encompassed by the present invention.

Monoclonal antibodies secreted by a hybridoma cell line can be purifiedusing any technique known in the art. Hybridomas or mAbs may be furtherscreened to identify mAbs with particular properties, such as theability to block an alpha4beta7 induced activity. Examples of suchscreens are provided in the examples below.

Monoclonal antibodies can also be produced using a process referred toas genetic immunization. For example, a nucleic acid encoding theantigen of interest can be incorporated into a viral vector (such as anadenoviral vector). The resulting vector is then used to develop animmune response against the antigen of interest in a suitable hostanimal (for example, a non-obese diabetic, or NOD, mouse). Thistechniques is substantially described by Ritter et al., Biodrugs 16(1):3-10 (2002), the disclosure of which is incorporated by referenceherein.

Molecular evolution of the complementarity determining regions (CDRs) inthe center of the antibody binding site also has been used to isolateantibodies with increased affinity, for example, antibodies havingincreased affinity for c-erbB-2, as described by Schier et al., 1996, J.Mol. Biol. 263:551. Accordingly, such techniques are useful in preparingantibodies to alpha4beta7.

Antigen binding proteins directed against an alpha4beta7 can be used,for example, in assays to detect the presence of alpha4beta7polypeptides or cells expressing alpha4beta7, either in vitro or invivo. The antigen binding proteins also may be employed in purifyingalpha4beta7 proteins by immunoaffinity chromatography. Those antigenbinding proteins that additionally can block the interaction of MAdCAM-1and alpha4beta7 may be used to inhibit a biological activity thatresults from such interaction. Blocking antigen binding proteins can beused in the methods of the present invention. Such antigen bindingproteins that function as alpha4beta7 antagonists may be employed intreating any alpha4beta7-induced condition, including but not limited toinflammatory conditions. In one embodiment, a human anti-alpha4beta7monoclonal antibody generated by procedures involving immunization oftransgenic mice is employed in treating such conditions.

Antigen binding proteins may be employed in an in vitro procedure, oradministered in vivo to inhibit an alpha4beta7-induced biologicalactivity. Disorders caused or exacerbated (directly or indirectly) byalpha4beta7 and its interaction with MAdCAM-1, examples of which areprovided herein, thus may be treated. In one embodiment, the presentinvention provides a therapeutic method comprising in vivoadministration of an alpha4beta7 blocking antigen binding protein to amammal in need thereof in an amount effective for reducing analpha4beta7-induced biological activity.

Antigen binding proteins of the invention include partially human andfully human monoclonal antibodies that inhibit a biological activity ofalpha4beta7. One embodiment is directed to a human monoclonal antibodythat at least partially blocks the interaction of human alpha4beta7 withMAdCAM-1. In one embodiment, the antibodies are generated by immunizinga transgenic mouse with an alpha4beta7 immunogen. In another embodiment,the immunogen is a human alpha4beta7 polypeptide (e.g., a celltransformed or transfected to express alpha4beta7, or a cell thatnaturally expresses alpha4beta7). Hybridoma cell lines derived from suchimmunized mice, wherein the hybridoma secretes a monoclonal antibodythat binds alpha4beta7, also are provided herein.

Although human, partially human, or humanized antibodies will besuitable for many applications, particularly those involvingadministration of the antibody to a human subject, other types ofantigen binding proteins will be suitable for certain applications. Thenon-human antibodies of the invention can be, for example, derived fromany antibody-producing animal, such as mouse, rat, rabbit, goat, donkey,or non-human primate (such as monkey (e.g., cynomologous or rhesusmonkey) or ape (e.g., chimpanzee)). Non-human antibodies of theinvention can be used, for example, in in vitro and cell-culture basedapplications, or any other application where an immune response to theantibody of the invention does not occur, is insignificant, can beprevented, is not a concern, or is desired. In one embodiment, anon-human antibody of the invention is administered to a non-humansubject. In another embodiment, the non-human antibody does not elicitan immune response in the non-human subject. In another embodiment, thenon-human antibody is from the same species as the non-human subject,e.g., a mouse antibody of the invention is administered to a mouse. Anantibody from a particular species can be made by, for example,immunizing an animal of that species with the desired immunogen (e.g.,cells expressing alph4beta7, or a soluble alpha4beta7 polypeptide) orusing an artificial system for generating antibodies of that species(e.g., a bacterial or phage display-based system for generatingantibodies of a particular species), or by converting an antibody fromone species into an antibody from another species by replacing, e.g.,the constant region of the antibody with a constant region from theother species, or by replacing one or more amino acid residues of theantibody so that it more closely resembles the sequence of an antibodyfrom the other species. In one embodiment, the antibody is a chimericantibody comprising amino acid sequences derived from antibodies fromtwo or more different species.

Antigen binding proteins may be prepared by any of a number ofconventional techniques. For example, they may be purified from cellsthat naturally express them (e.g., an antibody can be purified from ahybridoma that produces it), or produced in recombinant expressionsystems, using any technique known in the art. See, for example,Monoclonal Antibodies, Hybridomas: A New Dimension in BiologicalAnalyses, Kennet et al. (eds.), Plenum Press, New York (1980); andAntibodies: A Laboratory Manual, Harlow and Land (eds.), Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., (1988).

Any expression system known in the art can be used to make therecombinant polypeptides of the invention. In general, host cells aretransformed with a recombinant expression vector that comprises DNAencoding a desired polypeptide. Among the host cells that may beemployed are prokaryotes, yeast or higher eukaryotic cells. Prokaryotesinclude gram negative or gram positive organisms, for example E. coli orbacilli. Higher eukaryotic cells include insect cells and establishedcell lines of mammalian origin. Examples of suitable mammalian host celllines include the COS-7 line of monkey kidney cells (ATCC CRL 1651)(Gluzman et al., 1981, Cell 23:175), L cells, 293 cells, C127 cells, 3T3cells (ATCC CCL 163), Chinese hamster ovary (CHO) cells, HeLa cells, BHK(ATCC CRL 10) cell lines, and the CVI/EBNA cell line derived from theAfrican green monkey kidney cell line CVI (ATCC CCL 70) as described byMcMahan et al., 1991, EMBO J. 10: 2821. Appropriate cloning andexpression vectors for use with bacterial, fungal, yeast, and mammaliancellular hosts are described by Pouwels et al. (Cloning Vectors: ALaboratory Manual, Elsevier, N.Y., 1985).

The transformed cells can be cultured under conditions that promoteexpression of the polypeptide, and the polypeptide recovered byconventional protein purification procedures. One such purificationprocedure includes the use of affinity chromatography, e.g., over amatrix having all or a portion (e.g., the extracellular domain) ofalpha4beta7 bound thereto. Polypeptides contemplated for use hereininclude substantially homogeneous recombinant mammalian anti-alpha4beta7antibody polypeptides substantially free of contaminating endogenousmaterials.

The amino acid sequence of the polypeptides may be verified by any meansknown in the art, and may be identical to the sequences disclosed hereinin the Sequence Listing, or may differ from those sequences at one ormore amino acid residues as result of processing. For example, on all ora portion of the substantially homogenous polypeptides, a C-terminalamino acid from either the light chain or the heavy chain (or relevantsingle-chain molecule) may be removed, by proteolytic processing orother processing that occurs during culture, for example, processing ofC-terminal Lys residues. Alternatively, more than one C-terminal aminoacid residue is removed, for example two C-terminal amino acids, orthree, four or five C-terminal amino acids. For example, a C-terminalmight be truncated to amidated proline of the heavy chain of an antibodyas disclosed. Similarly, N-terminal amino acids may be absent, forexample, one, two, three, four or five N-terminal amino acids may beabsent.

Alternatively, or additionally, amino acid residues may undergopost-translational modifications, for example but not limited to,glutamine (in particular, glutamine at the N-terminus) may be cyclizedor converted to pyroglutamic acid; additionally or alternatively, aminoacids may undergo deamidation, isomerization, glycation and/oroxidation. The polypeptides of the invention may undergo additionalpost-translational modification, including glycosylation, for exampleN-linked or O-linked glycosylation, at sites that are well-known in theart. As described previously, changes may be made in the amino acidsequence of a polypeptide to preclude or minimize such alterations, orto facilitate them in circumstances where such processing is beneficial.

Preparations of substantially homogenous polypeptides may comprise about1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%,98%, or 99% polypeptide having undergone a particular form (or forms) ofprocessing. Preparations of substantially homogenous polypeptides maycomprise some (less than or equal to 50%), most (more than 50% but lessthan 90%) or substantially all (more than 90%) of a particular form(s)of processed polypeptide. Moreover, such preparations may comprisepolypeptides that have varying levels of more than one type ofprocessing-related modification, for example, a polypeptide may havesome, most or substantially all of a C-terminal lysine removed (forexample, the C-terminal lysine in SEQ ID NO: 72) and some, most orsubstantially all of an N-terminal amino acid converted to pyroglutamicacid (for example, any polypeptide shown in Table 1 and/or 2 or in theconsensus sequences).

Antigen binding proteins may be prepared, and screened for desiredproperties, by any of a number of known techniques. Certain of thetechniques involve isolating a nucleic acid encoding a polypeptide chain(or portion thereof) of an antigen binding protein of interest (e.g., ananti-alpha4beta7 antibody), and manipulating the nucleic acid throughrecombinant DNA technology. The nucleic acid may be fused to anothernucleic acid of interest, or altered (e.g., by mutagenesis or otherconventional techniques) to add, delete, or substitute one or more aminoacid residues, for example.

In one aspect, the present invention provides antigen-binding fragmentsof an anti-alpha4beta7 antibody of the invention. Such fragments canconsist entirely of antibody-derived sequences or can compriseadditional sequences. Examples of antigen-binding fragments include Fab,F(ab′)₂, single chain antibodies, diabodies, triabodies, tetrabodies,and domain antibodies. Other examples are provided in Lunde et al.,2002, Biochem. Soc. Trans. 30:500-06.

Single chain antibodies may be formed by linking heavy and light chainvariable domain (Fv region) fragments via an amino acid bridge (shortpeptide linker), resulting in a single polypeptide chain. Suchsingle-chain Fvs (scFvs) have been prepared by fusing DNA encoding apeptide linker between DNAs encoding the two variable domainpolypeptides (V_(L) and V_(H)). The resulting polypeptides can fold backon themselves to form antigen-binding monomers, or they can formmultimers (e.g., dimers, trimers, or tetramers), depending on the lengthof a flexible linker between the two variable domains (Kortt et al.,1997, Prot. Eng. 10:423; Kortt et al., 2001, Biomol. Eng. 18:95-108). Bycombining different V_(L) and V_(H)-comprising polypeptides, one canform multimeric scFvs that bind to different epitopes (Kriangkum et al.,2001, Biomol. Eng. 18:31-40). Techniques developed for the production ofsingle chain antibodies include those described in U.S. Pat. No.4,946,778; Bird, 1988, Science 242:423; Huston et al., 1988, Proc. Natl.Acad. Sci. USA 85:5879; Ward et al., 1989, Nature 334:544, de Graaf etal., 2002, Methods Mol. Biol. 178:379-87.

Antigen binding proteins (e.g., antibodies, antibody fragments, andantibody derivatives) of the invention can comprise any constant regionknown in the art. The light chain constant region can be, for example, akappa- or lambda-type light chain constant region, e.g., a human kappa-or lambda-type light chain constant region. The heavy chain constantregion can be, for example, an alpha-, delta-, epsilon-, gamma-, ormu-type heavy chain constant regions, e.g., a human alpha-, delta-,epsilon-, gamma-, or mu-type heavy chain constant region. In oneembodiment, the light or heavy chain constant region is a fragment,derivative, variant, or mutein of a naturally occurring constant region.

Techniques are known for deriving an antibody of a different subclass orisotype from an antibody of interest, i.e., subclass switching. Thus,IgG antibodies may be derived from an IgM antibody, for example, andvice versa. Such techniques allow the preparation of new antibodies thatpossess the antigen-binding properties of a given antibody (the parentantibody), but also exhibit biological properties associated with anantibody isotype or subclass different from that of the parent antibody.Recombinant DNA techniques may be employed. Cloned DNA encodingparticular antibody polypeptides may be employed in such procedures,e.g., DNA encoding the constant domain of an antibody of the desiredisotype. See also Lantto et al., 2002, Methods Mol. Biol. 178:303-16.Moreover, if an IgG4 is desired, it may also be desired to introduce apoint mutation (CPSCP->CPPCP) in the hinge region as described in Bloomet al., 1997, Protein Science 6:407, incorporated by reference herein)to alleviate a tendency to form intra-H chain disulfide bonds that canlead to heterogeneity in the IgG4 antibodies.

Moreover, techniques for deriving antigen binding proteins havingdifferent properties (i.e., varying affinities for the antigen to whichthey bind) are also known. One such technique, referred to as chainshuffling, involves displaying immunoglobulin variable domain generepertoires on the surface of filamentous bacteriophage, often referredto as phage display. Chain shuffling has been used to prepare highaffinity antibodies to the hapten 2-phenyloxazol-5-one, as described byMarks et al., 1992, BioTechnology, 10:779.

In another embodiment, the present invention provides an antigen bindingprotein that has a low dissociation constant from alpha4beta7. In oneembodiment, the antigen binding protein has a K_(d) of 100 pM or lower.In another embodiment, the K_(d) is 10 pM or lower; in anotherembodiment, it is 5 pM or lower, or it is 1 pM or lower. In anotherembodiment, the K_(d) is substantially the same as an antibody describedherein in the Examples. In another embodiment, the antigen bindingprotein binds to alpha4beta7 with substantially the same K_(d) as anantibody described herein in the Examples.

In another aspect, the present invention provides an antigen bindingprotein that inhibits an activity of alpha4beta7, for example binding(or adhesion) to MAdCAM-1, binding to cells expressing MAdCAM-1, oradhesion between cells expressing alpha4beta 7 and cells expressingMAdCAM-1. In one embodiment, the antigen binding protein has an IC₅₀ of1000 pM or lower. In another embodiment, the IC₅₀ is 500 pM or lower; inanother embodiment, the IC₅₀ is 100 pM or lower. In another embodiment,the IC₅₀ is substantially the same as that of an antibody describedherein in the Examples. In another embodiment, the antigen bindingprotein inhibits an activity of alpha4beta7 with substantially the sameIC₅₀ as an antibody described herein in the Examples.

In one embodiment, antigen binding proteins of the present inventionhave an apparent affinity for alpha4beta7 (or cells expressingalpha4beta7) of 1000 pM or lower. In other embodiments, the antigenbinding proteins exhibit an apparent affinity of 500 pM or lower, 200 pMor lower, 100 pM or lower, 80 pM or lower, 40 pM or lower, or 15 pM orlower. In another embodiment, the antigen binding protein exhibits anapparent affinity substantially the same as that of an antibodydescribed herein in the Examples. In another embodiment, the antigenbinding protein has an apparent affinity substantially the same that ofan antibody described herein in the Examples.

In another aspect, the present invention provides an antigen bindingprotein that binds both active and inactive forms of alpha4beta7. Inanother embodiment, an antigen binding protein binds only one form, orpreferentially binds one form, of alpha4beta7. For example, an antigenbinding protein may bind alpha4beta7 in the presence or absence of Mn²⁺(i.e., it binds both active and inactive forms). Alternatively, anantigen binding protein may bind alpha4beta7 only in the presence ofMn²⁺ or only in the absence of Mn²⁺, or it may bind with higher affinityunder one such condition than another, indicating preferential bindingto a particular form of alpha4beta7.

In another embodiment, the present invention provides an antigen bindingprotein that competes for binding to alpha4beta7 with an antibodydisclosed herein. Such competitive ability can be determined by methodsthat are well-known in the art, for example by competition in binding toalpha4beta7-expressing cells as observed using fluorescence activatecells sorting (FACS) techniques or other, similar assays, by competitionin an assay such as an adhesion assay (i.e., between cells expressingalpha4beta7 and cells expressing MAdCAM-1), or by competition in anotherassay described herein. In one aspect, an antigen binding protein thatcompetes for binding to alpha4beta7 with an antibody disclosed hereinbinds the same epitope or an overlapping (or adjacent) epitope as theantibody. In another aspect, the antigen binding protein that competesfor binding to alpha4beta7 with an antibody disclosed herein inhibits anactivity of alpha4beta7.

In another aspect, the present invention provides an antigen bindingprotein that binds to human alpha4beta7 expressed on the surface of acell and, when so bound, inhibits alpha4beta7 interaction with MAdCAM-1without causing a significant reduction in the amount of alpha4beta7 onthe surface of the cell. Any method for determining or estimating theamount of alpha4beta7 on the surface and/or in the interior of the cellcan be used. In one embodiment, the present invention provides anantigen binding protein that binds to alpha4beta7 expressed on thesurface of a cell and, when so bound, inhibits alpha4beta7 interactionwith MAdCAM-1 without significantly increasing the rate ofinternalization of the alpha4beta7 from the surface of the cell. Inother embodiments, binding of the antigen binding protein to thealpha4beta7-expressing cell causes less than about 75%, 50%, 40%, 30%,20%, 15%, 10%, 5%, 1%, or 0.1% of the cell-surface alpha4beta7 to beinternalized.

In another aspect, the present invention provides an antigen bindingprotein having a half-life of at least one day in vitro or in vivo(e.g., when administered to a human subject). In one embodiment, theantigen binding protein has a half-life of at least three days. Inanother embodiment, the antigen binding protein has a half-life of fourdays or longer. In another embodiment, the antigen binding protein has ahalf-life of eight days or longer. In another embodiment, the antigenbinding protein is derivatized or modified such that it has a longerhalf-life as compared to the underivatized or unmodified antigen bindingprotein. In another embodiment, the antigen binding protein contains oneor more point mutations to increase serum half life, such as describedin WO 00/09560, published Feb. 24, 2000, incorporated by reference.

The present invention further provides multi-specific antigen bindingproteins, for example, bispecific antigen binding protein, e.g., antigenbinding protein that bind to two different epitopes of alpha4beta7, orto an epitope of alpha4beta7 and an epitope of another molecule, via twodifferent antigen binding sites or regions. Moreover, bispecific antigenbinding protein as disclosed herein can comprise an alpha4beta7 bindingsite from one of the herein-described antibodies and a secondalpha4beta7 binding region from another of the herein-describedantibodies, including those described herein by reference to otherpublications. Alternatively, a bispecific antigen binding protein maycomprise an antigen binding site from one of the herein describedantibodies and a second antigen binding site from another alpha4beta7antibody that is known in the art, or from an antibody that is preparedby known methods or the methods described herein.

Numerous methods of preparing bispecific antibodies are known in theart, and discussed in U.S. patent application Ser. No. 09/839,632, filedApr. 20, 2001 (incorporated by reference herein). Such methods includethe use of hybrid-hybridomas as described by Milstein et al., 1983,Nature 305:537, and others (U.S. Pat. No. 4,474,893, U.S. Pat. No.6,106,833), and chemical coupling of antibody fragments (Brennan et al.,1985, Science 229:81; Glennie et al., 1987, J. Immunol. 139:2367; U.S.Pat. No. 6,010,902). Moreover, bispecific antibodies can be produced viarecombinant means, for example by using leucine zipper moieties (i.e.,from the Fos and Jun proteins, which preferentially form heterodimers;Kostelny et al., 1992, J. Immunol. 148:1547) or other lock and keyinteractive domain structures as described in U.S. Pat. No. 5,582,996.Additional useful techniques include those described in Kortt et al.,1997, supra; U.S. Pat. No. 5,959,083; and U.S. Pat. No. 5,807,706.

In another aspect, the antigen binding protein of the present inventioncomprises a derivative of an antibody. The derivatized antibody cancomprise any molecule or substance that imparts a desired property tothe antibody, such as increased half-life in a particular use. Thederivatized antibody can comprise, for example, a detectable (orlabeling) moiety (e.g., a radioactive, colorimetric, antigenic orenzymatic molecule, a detectable bead (such as a magnetic orelectrodense (e.g., gold) bead), or a molecule that binds to anothermolecule (e.g., biotin or streptavidin)), a therapeutic or diagnosticmoiety (e.g., a radioactive, cytotoxic, or pharmaceutically activemoiety), or a molecule that increases the suitability of the antibodyfor a particular use (e.g., administration to a subject, such as a humansubject, or other in vivo or in vitro uses). Examples of molecules thatcan be used to derivatize an antibody include albumin (e.g., human serumalbumin) and polyethylene glycol (PEG). Albumin-linked and PEGylatedderivatives of antibodies can be prepared using techniques well known inthe art. In one embodiment, the antibody is conjugated or otherwiselinked to transthyretin (TTR) or a TTR variant. The TTR or TTR variantcan be chemically modified with, for example, a chemical selected fromthe group consisting of dextran, poly(n-vinyl pyurrolidone),polyethylene glycols, propropylene glycol homopolymers, polypropyleneoxide/ethylene oxide co-polymers, polyoxyethylated polyols and polyvinylalcohols. US Pat. App. No. 20030195154.

In another aspect, the present invention provides methods of screeningfor a molecule that binds to alpha4beta7 using the antigen bindingproteins of the present invention. Any suitable screening technique canbe used. In one embodiment, an alpha4beta7 molecule, or a fragmentthereof to which an antigen binding protein of the present inventionbinds, is contacted with the antigen binding protein of the inventionand with another molecule, wherein the other molecule binds toalpha4beta7 if it reduces the binding of the antigen binding protein toalpha4beta7. Binding of the antigen binding protein can be detectedusing any suitable method, e.g., an ELISA. Detection of binding of theantigen binding protein to alpha4beta7 can be simplified by detectablylabeling the antigen binding protein, as discussed above. In anotherembodiment, the alpha4beta7-binding molecule is further analyzed todetermine whether it inhibits alpha4beta7 activation and/or signaling.

Nucleic Acids

In one aspect, the present invention provides isolated nucleic acidmolecules. The nucleic acids comprise, for example, polynucleotides thatencode all or part of an antigen binding protein, for example, one orboth chains of an antibody of the invention, or a fragment, derivative,mutein, or variant thereof, polynucleotides sufficient for use ashybridization probes, PCR primers or sequencing primers for identifying,analyzing, mutating or amplifying a polynucleotide encoding apolypeptide, anti-sense nucleic acids for inhibiting expression of apolynucleotide, and complementary sequences of the foregoing. Thenucleic acids can be any length. They can be, for example, 5, 10, 15,20, 25, 30, 35, 40, 45, 50, 75, 100, 125, 150, 175, 200, 250, 300, 350,400, 450, 500, 750, 1,000, 1,500, 3,000, 5,000 or more nucleotides inlength, and/or can comprise one or more additional sequences, forexample, regulatory sequences, and/or be part of a larger nucleic acid,for example, a vector. The nucleic acids can be single-stranded ordouble-stranded and can comprise RNA and/or DNA nucleotides, andartificial variants thereof (e.g., peptide nucleic acids).

Nucleic acids encoding antibody polypeptides (e.g., heavy or lightchain, variable domain only, or full length) may be isolated fromB-cells of mice that have been immunized with alpha4beta7. The nucleicacid may be isolated by conventional procedures such as polymerase chainreaction (PCR).

The invention further provides nucleic acids that hybridize to othernucleic acids under particular hybridization conditions. Methods forhybridizing nucleic acids are well-known in the art. See, e.g., CurrentProtocols in Molecular Biology, John Wiley & Sons, N.Y. (1989),6.3.1-6.3.6. As defined herein, a moderately stringent hybridizationcondition uses a prewashing solution containing 5× sodiumchloride/sodium citrate (SSC), 0.5% SDS, 1.0 mM EDTA (pH 8.0),hybridization buffer of about 50% formamide, 6×SSC, and a hybridizationtemperature of 55° C. (or other similar hybridization solutions, such asone containing about 50% formamide, with a hybridization temperature of42° C.), and washing conditions of 60° C., in 0.5×SSC, 0.1% SDS. Astringent hybridization condition hybridizes in 6×SSC at 45° C.,followed by one or more washes in 0.1×SSC, 0.2% SDS at 68° C.Furthermore, one of skill in the art can manipulate the hybridizationand/or washing conditions to increase or decrease the stringency ofhybridization such that nucleic acids comprising nucleotide sequencesthat are at least 65, 70, 75, 80, 85, 90, 95, 98 or 99% identical toeach other typically remain hybridized to each other. The basicparameters affecting the choice of hybridization conditions and guidancefor devising suitable conditions are set forth by, for example,Sambrook, Fritsch, and Maniatis (1989, Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,chapters 9 and 11; and Current Protocols in Molecular Biology, 1995,Ausubel et al., eds., John Wiley & Sons, Inc., sections 2.10 and6.3-6.4), and can be readily determined by those having ordinary skillin the art based on, for example, the length and/or base composition ofthe DNA.

Changes can be introduced by mutation into a nucleic acid, therebyleading to changes in the amino acid sequence of a polypeptide (e.g., anantigen binding protein) that it encodes. Mutations can be introducedusing any technique known in the art. In one embodiment, one or moreparticular amino acid residues are changed using, for example, asite-directed mutagenesis protocol. In another embodiment, one or morerandomly selected residues is changed using, for example, a randommutagenesis protocol. However it is made, a mutant polypeptide can beexpressed and screened for a desired property (e.g., binding toalpha4beta7 or blocking the binding of alpha4beta7 to an addressin suchas MAdCAM).

Mutations can be introduced into a nucleic acid without significantlyaltering the biological activity of a polypeptide that it encodes. Forexample, one can make nucleotide substitutions leading to amino acidsubstitutions at non-essential amino acid residues. In one embodiment, anucleotide sequence, or a desired fragment, variant, or derivativethereof, is mutated such that it encodes an amino acid sequencecomprising one or more deletions or substitutions of amino acidresidues. In another embodiment, the mutagenesis inserts an amino acidadjacent to one or more amino acid residues. Alternatively, one or moremutations can be introduced into a nucleic acid that selectively changethe biological activity (e.g., binding of alpha4beta7, inhibitingbinding of alpha4beta7 to an addressin such as MAdCAM, etc.) of apolypeptide that it encodes. For example, the mutation canquantitatively or qualitatively change the biological activity. Examplesof quantitative changes include increasing, reducing or eliminating theactivity. Examples of qualitative changes include changing the antigenspecificity of an antigen binding protein.

In another aspect, the present invention provides nucleic acid moleculesthat are suitable for use as primers or hybridization probes for thedetection of nucleic acid sequences of the invention. A nucleic acidmolecule of the invention can comprise only a portion of a nucleic acidsequence encoding a full-length polypeptide of the invention, forexample, a fragment that can be used as a probe or primer or a fragmentencoding an active portion (e.g., an alpha4beta7 binding portion) of apolypeptide of the invention.

Probes based on the sequence of a nucleic acid of the invention can beused to detect the nucleic acid or similar nucleic acids, for example,transcripts encoding a polypeptide of the invention. The probe cancomprise a label group, e.g., a radioisotope, a fluorescent compound, anenzyme, or an enzyme co-factor. Such probes can be used to identify acell that expresses the polypeptide.

In another aspect, the present invention provides vectors comprising anucleic acid encoding a polypeptide of the invention or a portionthereof. Examples of vectors include, but are not limited to, plasmids,viral vectors, non-episomal mammalian vectors and expression vectors,for example, recombinant expression vectors.

The recombinant expression vectors of the invention can comprise anucleic acid of the invention in a form suitable for expression of thenucleic acid in a host cell. The recombinant expression vectors includeone or more regulatory sequences, selected on the basis of the hostcells to be used for expression, which is operably linked to the nucleicacid sequence to be expressed. Regulatory sequences include those thatdirect constitutive expression of a nucleotide sequence in many types ofhost cells (e.g., SV40 early gene enhancer, Rous sarcoma virus promoterand cytomegalovirus promoter), those that direct expression of thenucleotide sequence only in certain host cells (e.g., tissue-specificregulatory sequences, see Voss et al., 1986, Trends Biochem. Sci.11:287, Maniatis et al., 1987, Science 236:1237, incorporated byreference herein in their entireties), and those that direct inducibleexpression of a nucleotide sequence in response to particular treatmentor condition (e.g., the metallothionin promoter in mammalian cells andthe tet-responsive and/or streptomycin responsive promoter in bothprokaryotic and eukaryotic systems (see id.). It will be appreciated bythose skilled in the art that the design of the expression vector candepend on such factors as the choice of the host cell to be transformed,the level of expression of protein desired, etc. The expression vectorsof the invention can be introduced into host cells to thereby produceproteins or peptides, including fusion proteins or peptides, encoded bynucleic acids as described herein.

In another aspect, the present invention provides host cells into whicha recombinant expression vector of the invention has been introduced. Ahost cell can be any prokaryotic cell (for example, E. coli) oreukaryotic cell (for example, yeast, insect, or mammalian cells (e.g.,CHO cells)). Vector DNA can be introduced into prokaryotic or eukaryoticcells via conventional transformation or transfection techniques. Forstable transfection of mammalian cells, it is known that, depending uponthe expression vector and transfection technique used, only a smallfraction of cells may integrate the foreign DNA into their genome. Inorder to identify and select these integrants, a gene that encodes aselectable marker (e.g., for resistance to antibiotics) is generallyintroduced into the host cells along with the gene of interest.Preferred selectable markers include those that confer resistance todrugs, such as G418, hygromycin and methotrexate. Cells stablytransfected with the introduced nucleic acid can be identified by drugselection (e.g., cells that have incorporated the selectable marker genewill survive, while the other cells die), among other methods.

Indications

In one aspect, the present invention provides methods of treating asubject. The method can, for example, have a generally salubrious effecton the subject, e.g., it can increase the subject's expected longevity.Alternatively, the method can, for example, treat, prevent, cure,relieve, or ameliorate (“treat”) a disease, disorder, condition, orillness (“a condition”). Among the conditions to be treated inaccordance with the present invention are conditions characterized byinappropriate expression or activity of alpha4beta7. Such conditionsinclude those that are associated with inappropriate trafficking ofcells, for example, the trafficking of leukocytes (such as lymphocytesor monocytes) to the gastrointestinal tract or other tissues comprisingcells that express MAdCAM-1 (as a result of binding of the leukocytes tothe cells that express MAdCAM-1). Diseases which can be treatedaccordingly include inflammatory bowel disease, such as ulcerativecolitis, Crohn's disease, Celiac disease (nontropical Sprue),enteropathy associated with seronegative arthropathies, microscopic orcollagenous colitis, eosinophilic gastroenteritis, or pouchitisresulting after proctocolectomy and ileoanal anastomosis. Additionalconditions that may be treated in accordance with the present inventioninclude pancreatitis, insulin-dependent diabetes mellitus, mastitis,cholecystitis, cholangitis, pericholangitis, chronic bronchitis, chronicsinusitis, asthma and graft versus host disease.

Therapeutic Methods and Administration of Antigen Binding Proteins

Certain methods provided herein comprise administering an alpha4beta7heterodimer specific antigen binding protein to a subject, therebyreducing an alpha4beta7-induced biological response that plays a role ina particular condition. In particular embodiments, methods of theinvention involve contacting endogenous alpha4beta7 with an alpha4beta7antigen binding protein, e.g., via administration to a subject or in anex vivo procedure.

The term “treatment” encompasses alleviation or prevention of at leastone symptom or other aspect of a disorder, or reduction of diseaseseverity, and the like. An antigen binding protein need not effect acomplete cure, or eradicate every symptom or manifestation of a disease,to constitute a viable therapeutic agent. As is recognized in thepertinent field, drugs employed as therapeutic agents may reduce theseverity of a given disease state, but need not abolish everymanifestation of the disease to be regarded as useful therapeuticagents. Similarly, a prophylactically administered treatment need not becompletely effective in preventing the onset of a condition in order toconstitute a viable prophylactic agent. Simply reducing the impact of adisease (for example, by reducing the number or severity of itssymptoms, or by increasing the effectiveness of another treatment, or byproducing another beneficial effect), or reducing the likelihood thatthe disease will occur or worsen in a subject, is sufficient. Oneembodiment of the invention is directed to a method comprisingadministering to a patient an alpha4beta7 antagonist in an amount andfor a time sufficient to induce a sustained improvement over baseline ofan indicator that reflects the severity of the particular disorder.

As is understood in the pertinent field, pharmaceutical compositionscomprising the molecules of the invention are administered to a subjectin a manner appropriate to the indication. Pharmaceutical compositionsmay be administered by any suitable technique, including but not limitedto parenterally, topically, or by inhalation. If injected, thepharmaceutical composition can be administered, for example, viaintra-articular, intravenous, intramuscular, intralesional,intraperitoneal or subcutaneous routes, by bolus injection, orcontinuous infusion. Localized administration, e.g. at a site of diseaseor injury is contemplated, as are transdermal delivery and sustainedrelease from implants. Delivery by inhalation includes, for example,nasal or oral inhalation, use of a nebulizer, inhalation of theantagonist in aerosol form, and the like. Other alternatives includeeyedrops; oral preparations including pills, syrups, lozenges or chewinggum; and topical preparations such as lotions, gels, sprays, andointments.

Use of antigen binding proteins in ex vivo procedures also iscontemplated. For example, a patient's blood or other bodily fluid maybe contacted with an antigen binding protein that binds alpha4beta7 exvivo. The antigen binding protein may be bound to a suitable insolublematrix or solid support material.

Advantageously, antigen binding proteins are administered in the form ofa composition comprising one or more additional components such as aphysiologically acceptable carrier, excipient or diluent. Optionally,the composition additionally comprises one or more physiologicallyactive agents, for example, a second inflammation- or immune-inhibitingsubstance, an anti-angiogenic substance, an analgesic substance, etc.,non-exclusive examples of which are provided herein. In variousparticular embodiments, the composition comprises one, two, three, four,five, or six physiologically active agents in addition to an alpha4beta7binding antigen binding protein

In one embodiment, the pharmaceutical composition comprise an antigenbinding protein of the invention together with one or more substancesselected from the group consisting of a buffer, an antioxidant such asascorbic acid, a low molecular weight polypeptide (such as those havingfewer than 10 amino acids), a protein, an amino acid, a carbohydratesuch as glucose, sucrose or dextrins, a chelating agent such as EDTA,glutathione, a stabilizer, and an excipient. Neutral buffered saline orsaline mixed with conspecific serum albumin are examples of appropriatediluents. In accordance with appropriate industry standards,preservatives such as benzyl alcohol may also be added. The compositionmay be formulated as a lyophilizate using appropriate excipientsolutions (e.g., sucrose) as diluents. Suitable components are nontoxicto recipients at the dosages and concentrations employed. Furtherexamples of components that may be employed in pharmaceuticalformulations are presented in Remington's Pharmaceutical Sciences,16^(th) Ed. (1980) and 20^(th) Ed. (2000), Mack Publishing Company,Easton, Pa.

Kits for use by medical practitioners include an alpha4beta7-inhibitingsubstance of the invention and a label or other instructions for use intreating any of the conditions discussed herein. In one embodiment, thekit includes a sterile preparation of one or more alpha4beta7 bindingantigen binding proteins, which may be in the form of a composition asdisclosed above, and may be in one or more vials.

Dosages and the frequency of administration may vary according to suchfactors as the route of administration, the particular antigen bindingproteins employed, the nature and severity of the disease to be treated,whether the condition is acute or chronic, and the size and generalcondition of the subject. Appropriate dosages can be determined byprocedures known in the pertinent art, e.g. in clinical trials that mayinvolve dose escalation studies.

An alpha4beta7 inhibiting substance of the invention may beadministered, for example, once or more than once, e.g., at regularintervals over a period of time. In particular embodiments, an antigenbinding protein is administered over a period of at least a month ormore, e.g., for one, two, or three months or even indefinitely. Fortreating chronic conditions, long-term treatment is generally mosteffective. However, for treating acute conditions, administration forshorter periods, e.g. from one to six weeks, may be sufficient. Ingeneral, the antigen binding protein is administered until the patientmanifests a medically relevant degree of improvement over baseline forthe chosen indicator or indicators.

Particular embodiments of the present invention involve administering anantigen binding protein at a dosage of from about 1 ng of antigenbinding protein per kg of subject's weight per day (“1 ng/kg/day”) toabout 10 mg/kg/day, more preferably from about 500 ng/kg/day to about 5mg/kg/day, and most preferably from about 5 μg/kg/day to about 2mg/kg/day, to a subject. In additional embodiments, an antigen bindingprotein is administered to adults one time per week, two times per week,or three or more times per week, to treat an alpha4beta7 mediateddisease, condition or disorder, e.g., a medical disorder disclosedherein. If injected, the effective amount of antigen binding protein peradult dose may range from 1-20 mg/m², and preferably is about 5-12mg/m². Alternatively, a flat dose may be administered; the amount mayrange from 5-100 mg/dose. One range for a flat dose is about 20-30 mgper dose. In one embodiment of the invention, a flat dose of 25 mg/doseis repeatedly administered by injection. If a route of administrationother than injection is used, the dose is appropriately adjusted inaccordance with standard medical practices. One example of a therapeuticregimen involves injecting a dose of about 20-30 mg of antigen bindingprotein to one to three times per week over a period of at least threeweeks, though treatment for longer periods may be necessary to inducethe desired degree of improvement. For pediatric subjects (age 4-17),one exemplary suitable regimen involves the subcutaneous injection of0.4 mg/kg, up to a maximum dose of 25 mg of antigen binding proteinadministered two or three times per week.

Particular embodiments of the methods provided herein involvesubcutaneous injection of from 0.5 mg to 10 mg, preferably from 3 to 5mg, of an antigen binding protein, once or twice per week. Anotherembodiment is directed to pulmonary administration (e.g., by nebulizer)of 3 or more mg of antigen binding protein once a week.

Examples of therapeutic regimens provided herein comprise subcutaneousinjection of an antigen binding protein once a week, at a dose of 1.5 to3 mg, to treat a condition in which alpha4beta7 plays a role. Examplesof such conditions are provided herein and include, for example,rheumatic conditions as previously described, and other conditions inwhich excessive or inappropriate trafficking of alpha4beta7-expressingcells plays a role (described herein; for example, inflammatory boweldisease, pancreatitis, etc). Weekly administration of antigen bindingprotein is continued until a desired result is achieved, e.g., thesubject's symptoms subside. Treatment may resume as needed, or,alternatively, maintenance doses may be administered.

Other examples of therapeutic regimens provided herein comprisesubcutaneous or intravenous administration of a dose of 1, 3, 5, 6, 7,8, 9, 10, 11, 12, 15, or 20 milligrams of an alpha4beta7 inhibitor ofthe present invention per kilogram body mass of the subject (mg/kg). Thedose can be administered once to the subject, or more than once at acertain interval, for example, once a day, three times a week, twice aweek, once a week, three times a month, twice a month, once a month,once every two months, once every three months, once every six months,or once a year. The duration of the treatment, and any changes to thedose and/or frequency of treatment, can be altered or varied during thecourse of treatment in order to meet the particular needs of thesubject.

In another embodiment, an antigen binding protein is administered to thesubject in an amount and for a time sufficient to induce an improvement,preferably a sustained improvement, in at least one indicator thatreflects the severity of the disorder that is being treated. Variousindicators that reflect the extent of the subject's illness, disease orcondition may be assessed for determining whether the amount and time ofthe treatment is sufficient. Such indicators include, for example,clinically recognized indicators of disease severity, symptoms, ormanifestations of the disorder in question. In one embodiment, animprovement is considered to be sustained if the subject exhibits theimprovement on at least two occasions separated by two to four weeks.The degree of improvement generally is determined by a physician, whomay make this determination based on signs, symptoms, biopsies, or othertest results, and who may also employ questionnaires that areadministered to the subject, such as quality-of-life questionnairesdeveloped for a given disease.

Alteration of alpha4beta7 expression and/or activation of alpha4beta7,and or its binding partner MAdCAM-1, are associated with a number ofdisorders, including, for example, inflammatory conditions of thegastrointestinal system. Subjects with a given disorder may be screened,to identify those individuals who have altered alpha4beta7 or MAdCAM-1expression and/or activation, thereby identifying the subjects who maybenefit most from treatment with an alpha4beta7 binding antigen bindingprotein. Thus, treatment methods provided herein optionally comprise afirst step of measuring a subject's alpha4beta7 or MAdCAM-1 activationor expression levels. An antigen binding protein may be administered toa subject in whom alpha4beta7 and/or MAdCAM-1 expression and/oractivation is elevated above normal.

A subject's levels of alpha4beta7 or MAdCAM-1 activity may be monitoredbefore, during and/or after treatment with an antigen binding protein,to detect changes, if any, in alpha4beta7 or MAdCAM-1 activity. For somedisorders, the incidence of elevated alpha4beta7 and/or MAdCAM-1activity may vary according to such factors as the stage of the diseaseor the particular form of the disease. Known techniques may be employedfor measuring such activity, e.g., in a subject's blood or tissuesamples. Alpha4beta7 or MAdCAM1 activity may be measured using anysuitable technique.

Particular embodiments of methods and compositions of the inventioninvolve the use of an antigen binding protein and one or more additionalalpha4beta7 antagonists, for example, two or more antigen bindingproteins of the invention, or an antigen binding protein of theinvention and one or more other alpha4beta7 antagonists. In furtherembodiments, antigen binding protein are administered alone or incombination with other agents useful for treating the condition withwhich the patient is afflicted. Examples of such agents include bothproteinaceous and non-proteinaceous drugs. When multiple therapeuticsare co-administered, dosages may be adjusted accordingly, as isrecognized in the pertinent art. “Co-administration” and combinationtherapy are not limited to simultaneous administration, but also includetreatment regimens in which an antigen binding protein is administeredat least once during a course of treatment that involves administeringat least one other therapeutic agent to the patient.

Examples of other agents that may be co-administered with an antigenbinding protein are other antigen binding proteins or therapeuticpolypeptides that are chosen according to the particular condition to betreated. Alternatively, non-proteinaceous drugs that are useful intreating one of the particular conditions discussed above may beco-administered with an alpha4beta7 antagonist.

Combination Therapy

In another aspect, the present invention provides a method of treating asubject with an alpha4beta7 inhibiting antigen binding protein and oneor more other treatments. In one embodiment, such a combination therapyachieves synergy or an additive effect by, for example, attackingmultiple sites or molecular targets in a tumor. Types of combinationtherapies that can be used in connection with the present inventioninclude inhibiting or activating (as appropriate) multiple nodes in asingle disease-related pathway, multiple pathways in a target cell, andmultiple cell types within a target tissue.

In another embodiment, a combination therapy method comprisesadministering to the subject two, three, four, five, six, or more of thealpha4beta7 agonists or antagonists described herein. In anotherembodiment, the method comprises administering to the subject two ormore treatments that together inhibit or activate (directly orindirectly) alpha4beta7-mediated signal transduction. Examples of suchmethods include using combinations of two or more alpha4beta7 inhibitingantigen binding proteins, of an alpha4beta7 inhibiting antigen bindingprotein and one or more other therapeutic moiety havinganti-inflammatory properties (for example, non-steroidalanti-inflammatory agents, steroids, and/or immunomodulators), or of analpha4beta7 inhibiting antigen binding protein and one or more othertreatments (e.g., surgery, ultrasound, or treatment effective to reduceinflammation). Useful agents that may be combined with alpha4beta7inhibitors include those used to treat, for example, Crohn's disease orulcerative colitis, such as aminosalicylate (for example, mesalamine),corticosteroids (including predisone), antibiotics such as metronidazoleor ciprofloxacin (or other antibiotics useful for treating, for example,patients afflicted with fistulas), and immunosupporessives such asazathioprine, 6-mercaptopurine, methotrexate, tacrolimus andcyclosporine. Combinations of such agents are also contemplated for usewith the inventive alpha4beta7 inhibitors. Such agent(s) may beadministered orally or by another route, for example via suppository orenema.

Furthermore, one or more anti-alpha4beta7 antibodies or antibodyderivatives can be used in combination with one or more molecules orother treatments, wherein the other molecule(s) and/or treatment(s) donot directly bind to or affect alpha4beta7, but which combination iseffective for treating or preventing the condition being treated. Forexample, an alpha4eta7 inhibitor can be used in combination withprobiotic therapy, or other therapy used to restore or maintain normalgut flora. In one embodiment, one or more of the molecule(s) and/ortreatment(s) treats or prevents a condition that is caused by one ormore of the other molecule(s) or treatment(s) in the course of therapy,e.g., nausea, fatigue, alopecia, cachexia, insomnia, etc. In every casewhere a combination of molecules and/or other treatments is used, theindividual molecule(s) and/or treatment(s) can be administered in anyorder, over any length of time, which is effective, e.g.,simultaneously, consecutively, or alternately. In one embodiment, themethod of treatment comprises completing a first course of treatmentwith one molecule or other treatment before beginning a second course oftreatment. The length of time between the end of the first course oftreatment and beginning of the second course of treatment can be anylength of time that allows the total course of therapy to be effective,e.g., seconds, minutes, hours, days, weeks, months, or even years.

In another embodiment, the method comprises administering one or more ofthe alpha4beta7 antagonists described herein and one or more othertreatments (e.g., a therapeutic or palliative treatment). Where a methodcomprises administering more than one treatment to a subject, it is tobe understood that the order, timing, number, concentration, and volumeof the administrations is limited only by the medical requirements andlimitations of the treatment, i.e., two treatments can be administeredto the subject, e.g., simultaneously, consecutively, alternately, oraccording to any other regimen.

The following examples, both actual and prophetic, are provided for thepurpose of illustrating specific embodiments or features of the instantinvention and do not limit its scope.

EXAMPLE 1 Preparation of Antibodies

Monoclonal antibodies against human alpha4beta7 were developed byimmunizing XenoMouse™ XG2kappalambda (kl) and XG4kl mice (transgenicmice that express human IgG2 or IgG4, and human kappa and lambda lightchains, respectively; Abgenix Inc., Fremont Calif.) with cellsexpressing human alpha4beta7, either transiently transfected humanembryonic kidney (HEK) 293 cells (293-a4b7) or stably transfectedChinese hamster ovary (CHO) cells (CHO-a4b7). Serum titer was monitoredby fluorescence activated cell sorter (FACS) analysis comparingalpha4beta7 transfected cells to the respective parental control cells.Hyperimmune animals from either immunization campaign were sacrificedand spleen and lymph node tissues were subjected to hybridoma fusion.

Alpha4beta7 heterodimer specific antibodies were identified using aseries of assays. Hybridoma supernatants were first screened byFluorometric Microvolume Assay Technology (FMAT™ Applera Corporation,Foster City Calif.; a high-throughput screening cellular detectionsystem) for binding to alpha4beta7 transfected cells as compared tomock-transfected cells. Supernatants identified as positive for bindingto alpha4beta7 (1001 positive binding supernatants from CHO-a4b7 cellimmunization campaign and 1143 positive binding supernatants from293-a4b7 cell immunization campaign) were evaluated for the ability toinhibit HUT78 cell adhesion to MAdCAM-1-Fc in a similar fashion asdescribed (Erle, J. Immunol, (1994) 153:517). In this assay, 60supernatants from CHO-a4b7 campaign and 174 supernatants from 293-a4b7campaign showed greater than 90% inhibition (n=2) and were subject tofurther specificity and potency analysis.

Alpha4beta7-transfected, alpha4beta1-transfected, andalphaEbeta7-transfected 293 cells were prepared and used in FACSanalysis with the hybridoma supernatants that were identified in theinhibition assay. Supernatants that demonstrated binding to only thealpha4beta7 transfected cells were classified as heterodimers-specific,since antibodies to the alpha4 subunit of this integrin would also bindthe alpha4beta1-transfected cells, and antibodies that bound the beta7chain would bind alphaEbeta7-transfected cells. The hybridomasupernatants were also analyzed for binding activity to cynomologousmonkey alpha4beta7-transfected 293 cells by FACS analysis. Seven linesfrom the CHO-a4b7 campaign and 25 lines from the 293-a4b7 campaign wereselected for sub-cloning and further analysis.

EXAMPLE 2 Analysis of Antibodies

The antibody-secreting cells obtained were cloned, and theantibody-encoding nucleic acids were isolated and sequenced. Sitedirected mutagenesis was used to prepared variants that differed fromthe isolated sequences at one or more amino acid residues. The aminoacid sequence of the light and heavy chains of the antibodies andvariants are shown in Tables 1 and 2 below. It is recognized that theboundaries of the CDR and FR regions can vary from that shown below, asdiscussed previously herein.

TABLE 1 Sequence analysis of light chains Light chain FR1 CDR1 FR2 CDR21A10K DIQMTQSPSSVSASVGDRVTITC RASQGVSSWLA WYQQKPGMAPKLLIY AASILQS 11E7K1EIVMTQSPATLSVSPGETATLSC RASQTVSSNLA WYQQKPGQAPRLLIY GASTRAT 11E7K2DIQMTQSPSSLSASIGDRVTITC RASQGIRNYLA WYQRKPGKVPKLLIY AASTLQS 2F12KDIQMTQSPSSVFASVGDRVTITC RASQGISSWLA WYQQKPGKAPNLLIY GASSLQN 14E4LQSVLTQPPSVSAAPGQKVTISC SGSSSNIGNNYVS WYQQLPGTAPKLLIY DNNKRPS 3A5KDIQMTQSPSSVSASVGDRVTITC RASQGVISWLA WYQQKPGMAPKLLIY AASILQS 10D7KDIQMTQSPSSVSASVGDRVTITC RASQGVNNWLA WYQQKPGKAPKLLIF ATSSLQS 27D8KEIVMMQSPATLSVSPGERATLSC RASQSVSTNLA WYQQKPGQAPRLLIY GASTRAT 18A11KDIQMTQSPSSVSASVGDRVTITC RASQGISSWLA WYQQKPGKAPKLLIY GASNLES 20D7KEIVLTQSPGTLSLSPGERATLSC RASQSVSSSYLA WYQQKPGQAPRLLIY GASSRAT 23H6KEIVMTQSPATLSVSPGERATLSC RASQSVNSNLA WYQQKPGQAPRLLIY GASTRAT 27G8LQSVLTQPPSVSEAPRQRVTISC SGSNSNIGNNPVN WYQLFPGRAPKLLIY HDDLLPS 26C7KEIVMTQSPATLSVSPGERATLSC RASQSVSDNLA WYQQKPGQPPRLLIY GASTRAT 26H3KDIQMTQSPSSLSASVGDRVTITC QASQDISNYLN WYQQKPGKAPKLLIY DASNLET 19G6KDIQMTQSPSSLSASVGDRVTISC QASQDINTYLN WYQQKPGKVPKLLIY DASNLET 22B2KDVQMTQSPSSLSASVGDRVTITC QASQDITDYLN WYQQKPGKAPKLLIY DTSNLEA 24A2KEVMMTQSPATLSVSPGERATLSC RASQSVSSNLA WYQQKPGQAPRLLIF GASTRAT 26E9KELVMTQSPATLSVSPGERATVSC RASQSVSSDLA WYQQKPGQAPRLLIY GASSRAT 22F5KEIVMTQSPATLSVFPGEGATLSC RASQSVSSDLA WYQQKPGQAPRLLIY GASARAT 26C10KEIVLTQSPGTLSLSPGEGATLSC RASQTVTSSYLA WYQQSPSQSPRLLIY GASTRAT 17C8KEIVMTQSPATLSVSPGERATLSC RASQSVSSNLV WYQQKPGQAPRLLIY GASTRAT 25C9kDIQMTQSPSSVSASVGDRVTITC RASQDISSWLA WYQRKPGKAPKVLIY SASSLQS 19E6LSYELTQPPSVSVSPGQTASITC SGDKLGDKYAC WYQQKPGQSPVLVIY QDSKRPS 26G2kDIQMTQSPSSVSASVGDRVTITC RASQDISSWLA WYQQKPGTAPKVLIY SASSLQN 27G8L (a)QSVLTQPPSVSGAPRQRVTISC SGSNSNIGNNPVN WYQLFPGRAPKLLIY HDDLLPS 27G8L (b)QSVLTQPRSVSGAPRQRVTISC SGSNSNIGNNPVN WYQLFPGRAPKLLIY HDDLLPS 26H3K (c)DIQMTQSPSSLSASVGDRVTITC QASQDISNYLN WYQQKPGKAPKLLIY DASNLET 1A10K (d)DIQMTQSPSSVSASVGDRVTITC RASQGVSSWLA WYQQKPGKAPKLLIY AASTLQS Light chainFR3 CDR3 FR4 1A10K GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQANSFPWT FGQGTKVEIK11E7K1 GIPARFSGSGSGTEFTLTISSLQSEDFAVYYC QQYDYWPPLT FGGGTRVEIK 11E7K2GVPSRFSGSGSGTDFTLTISSLQPEDVATYCC QKYDSAPFT FGPGTKVDIK 2F12KGVPLRFSGSGSGTDFTLTISSLQPEDFATYYC QQANSFPWT FGQGTKVEIK 14E4LGIPDRFSGSKSGTSAILDITGLQTGDEADYYC GTWDSSLSAGRV FGGGTKLTVL 3A5KGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQANSFPWT FGQGTNVEIK 10D7KGVPSRFSGSGSGTDFTLTINSLQPEDFATYYC QQVNSFPGT FGQGTKVEIK 27D8KGIPARFSGSGSGTEFTLTISSLQSEDFAVYFC QQYNDWPT FGGGTKVEIK 18A11KGVPSRFSGSGSGTDFTLTISSLQPEDFANYYC QQANSFPWT FGQGTKVEIK 20D7KGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYC QQYDSSPPT FGGGTKVAIK 23H6KGIPARFSGSGSGTEFTLTISSLQSEDFAVYYC QQYDDWPPVT FGQGTRLEIK 27G8LGVSDRFSGSRSGTSASLAISGLQSEDETDYYC TAWDDSLNGWV FGGGTKLTVL 26C7KGIPARFSGSGSGTEFTLTISSLQSEDFAVYYC QQYDDWPT FGGGTRVEIK 26H3KGVPSRFSGSGSGTDFTFTINSLQPEDIATYFC QQYDNLPCS FGQGTKLEIK 19G6KGVPSRFSGSGSGTDFTFTISGLQPEDIATYYC QQFDNLPIT FGQGTRLEIK 22B2KGVPSRFSGSGSGTDFTFTISSLQPEDIATYYC QQYDILPYS FGQGTDLEIK 24A2KGIPARFSGSGSGTEFTLTISSLQSEDFAVYCC QQYDDWPT FGGGTKVEIK 26E9KGIPARFSGSGSGTEFTLTISSLQSEDFAVYYC QQYNNWPPLT FGGGTKVEIK 22F5KGIPARFSGSGSGTEFTLTISSLQSEDFAVYYC QQYHDWPPLS FGGGTKVEIK 26C10KGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYC QQYDSSPPT FGGGTKVEIK 17C8KGIPARFSGSGSGTDFTLTISSLQSEDFAVYYC QQYDDWPPLT FGGGTTVEIK 25C9kGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQADSFPWT FGQGTKVEIK 19E6LGIPERFSGSNSGNTATLTISGTQAMDEADYYC QAWDSSTVV FGGGTKLTVL 26G2kGVPSRFSGRGSGTDFALTISSLQPEDFATYYC QQADSFPWT FGRGTKVEIK 27G8L (a)GVSDRFSGSRSGTSASLAISGLQSADETDYYC TAWDDSLNGWV FGGGTKLTVL 27G8L (b)GVSDRFSGSRSGTSASLAISGLRSADETDYYC TAWDDSLNGWV FGGGTKLTVL 26H3K (c)GVPSRFSGSGSGTDFTFTINSLQPEDIATYFC QQYDNLPSS FGQGTKLEIK 1A10K (d)GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQANSFPWT FGQGTKVEIK

TABLE 2 Sequence analysis of heavy chains Heavy chain FR1 CDR1 FR2 CDR21A10H QVQLVQSGAEVKKPGASVKVSCKVSGYTLN DLSMH WVRQAPGKGLEWMGGFDPAEGKIISAQKFQD 11E7H1 QVQLVESGGGLVKPGGSLRLSCVASGFTFS DYYMSWIRQAPGKGLEWVS YISSSGSAIYYADSVKG 11E7H2 QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMH WVRQAPGKGLEWVA VIWYDGSNKYYADSVKG 2F12HQVQLVQSGAEVKKPGASVKVSCKVSGYTVT DLSMH WVRQAPGKGLEWMG GFDPQDGETIYAQKFQG14E4H QVQLVESGGGLVKPGGSLRLSCAASGFTFS DYYMS WIRQAPGKGLEWVSYISNSGSVVYYADSVKG 3A5H QVQLVQSGAEVKKPGASVKVSCKVSGYTLN DLSMHWVRQAPGKGLEWMG GFDPAEGKIISAQKFQD 10D7H QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMS WIRQAPGKGLEWVS YISSTGSAMYDADSVKG 27D8HQVQLVESGGGLVKPGGSLRLSCAASGFTFS DNYMS WIRQAPGKGLEWVS YISSSGSATYYADSVKG18A11H QVQLVQSGAEVKKPGASVKVSCKVSGYTLS DLSIH WVRQAPGKGLEWMGGFDPQDGETIYAQKFQG 20D7H QVQLVESGGGLVKPGGSLRLSCTASGFTFS DYYMSWIRQAPGKGLEWVS YISSSGSAIYYADSVKG 23H6H QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMS WIRQAPGKGLEWVS YISSSGSAMYSADSVKG 26G2HQVQLVESGGGLVKPGGSLRLSCAASGFTFS DYYMS WIRQAPGKGLEWVS YISSIGSAIHYADSVKG27G8H EVQLVESGGGLVQPGGSLRLSCAASGFTFS SYWMS WVRQASGKGLEWVANIKQDGSEKYYVDSVKG 26C7H QVQLVESGGGLVKPGGSLRLSCAASGFTFS DYYMSWIRQAPGKGLEWVS YISRVGSTTYYADSVKG 26H3H EVQLVQSGAEVKKPGESLKISCKGSGYSFTGYWIG WVRQMPGKGLEWMG IIYPYDSDTRYSPSFQG 19G6HQVQLVESGGDLVKPGGSLRLSCAASGFTFS DYYMS WIRQAPGKGLEWIS YISSSGSTMYYADSVKG22B2H EVQLVQSGAEVKEPGESLKISCKGSGYIFT SYWIA WVRQLPGKGLEWMGIIDPNDSDTRYSPSFQG 24A2H QVQLVESGGDLVEPGGSLRLSCAASGFTFR DYYMSWIRQAPGKGLEWVS YISSSGSAIYYADSVKG 26E9H QVQLVESGGGLVKPGGSLRLSCAASGFTFRDYYMS WIRQAPGKGLEWVS YISSSGSTSYCADSVKG 19E6HEVQLLESGGGLVQPGGSLRLSCAASGFTFS SYAMS WVRQAPGKGLEWVS AISGSGGSTYYADSVKG22F5H QVQLVESGGGLVKPGGSLRLSCAASGFTFS DYYMS WIRQAPGKGLEWVSYISSTGSTLYYADSVKG 25C9H QVQLVESGGGLVKPGGSLRLSCAASGFTFN DYYMSWIRQAPGKGLEWVS YISSSGSAIHYADSVKG 26C10H QVQLVESGGGLVKPGGSLRLSCVASGFTFSDYYMS WIRQTPGKGLEWVS YISSSGSAIHYADSVKG 17C8HQVQLVESGGGLVKPGGSLRLSCAASGFTFS DYYMS WIRQAPGKGLEWLS YISNSGSAMYYADSVKG1A10H(a) QVQLVQSGAEVKKPGASVKVSCKVSGYTLN DLSMH WVRQAPGKGLEWMGGFDPAEGKIISAQKFQD 27G8H(b) EVQLVESGGGLVKPGRSLRLSCAASGFTFS SYWMSWVRQASGKGLEWVA NIKQDGSEKYYVDSVKG Heavy chain FR3 CDR3 FR4 1A10HRVTMTDDTSTDTAYMELSSLRSEDSAVYYCAT LDFSSWFDP WGQGTLVTVSS 11E7H1RFTISRDNAKNSLYLQLNSLRAEDTAVYYCAR DYSSGWFYFDY WGRGTLVTVSS 11E7H2RFTISRDNSKNTLHLQMNSLRAEDTAVYYCAR EHWNYAFDI WGQGTMVTVSS 2F12HRVTMTEDTSTDTAYMELRSLRSEDTAVYYCTT ESSSAWFDP WGQGTLVTVSS 14E4HRFTISRHNAKNSLYLQMNSLRADDTAVYYCAR DRSSAWDEAFDI WGQGTMVTVSS 3A5HRVTMTDDTSTDTAYMELSSLRSEDSAVYYCAT LDFSSWFDP WGQGTLVTVSS 10D7HRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR EFSSGWSYFDY WGQGTLVTVSS 27D8HRFTISRDNAKNSLYLQMSSLRAEDTAVYYCAR DYSSGWYYFDY WGQGTLVTVSS 18A11HRVTMTEDTSTDTAYMELSSLKSEDTAVYYCAT GSSSSWFDP WGQGTLVTVSS 20D7HRFTISRDNAKNSLYLQMDSLRAEDTAVFYCAR EHSSGYWYFDL WGRGALVTVSS 23H6HRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR EYSSGWYYFDY WGRGTLVTVSS 26G2HRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR EYSSGWAYFDY WGQGTLVTVSS 27G8HRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR EGGYDWNYADYYGMDV WGQGTTVTVSS 26C7HRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR DYSSGWYYFDY WGQGTLVTVSS 26H3HQVTISADKSINTAYLQWSSLKASDTAMFYCAS HRLWLGEFPGPLNI WGQGTMVTVSS 19G6HRFTISRVNAKNSLYLQMNSLRAEDTAVYYCAR DRSSGLVSFDY WGQGTLVTVSS 22B2HQVTISADKSIHTAYLQWSSLKASDTAMYYCAT HRLWLGTLPGGFYI WGQGTMVTVSS 24A2HRFTISRDNPKNSLYLQMNSLRAEDTAVYYCAR DFSSGYYYFDY WGHGTLVTVSS 26E9HRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR DYSSGWFYFDY WGQGTLVTVSS 19E6HRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK APYSSSWALGLGMDV WGQGTTVTVSS 22F5HRFTISRDNAKNSLYLQMDSLRADDAAVYYCTR EYSSGWFFFDY WGQGTLVTVSS 25C9HRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR EYSSGWAYFDY WGQGTLVTVSS 26C10HRFTISRDNAKNSLYLQMDSLRAEDTAVFYCAR DHSSGYWYFDL WGRGTLVTVSS 17C8HRFTISRDNARNSLYLQMNSLRAEDTAVYYCAR EYSSGWFFFES WGQGTLVTVSS 1A10H(a)RVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR LDFSSWFDP WGQGTLVTVSS 27G8H(b)RFTISRDNAKNSLYLQMNSLRAGDTAVYYCAR EGGYDWNYADYYGMDV WGQGTTVTVSS

The amino acid sequences of the antibodies were further analyzed forsimilarities. The kappa light chains were grouped into three groups, anda consensus sequence was developed for each group. There were threeantibodies with lambda light chains, none of which bore enoughsimilarity to each other to form a group of related sequences from whicha consensus sequence could be developed. Two of the variants developedvaried in the lambda light chain. The heavy chains were grouped intofour groups with a single heavy chain categorized into a fifth group,and a consensus sequence was developed for groups 1 through 4. Theseresults are shown in Table 3(a) and 3(b) below; consensus sequences areshown in the Sequence Listing. The numbers in parentheses indicate theSEQ ID NO in the Sequence Listing.

TABLE 3(a) Grouping of antibodies by kappa light chain, withcorresponding heavy chain Kappa Heavy chain Kappa Heavy chain KappaHeavy chain Group 1 group Group 2 group Group 3 group (10 members)(H1-H5) (9 members) (H1-H5) (4 members) (H1-H5) 20D7K (10) H1 (38)11E7K2 (3) H1 (31) 22B2K (16) H4 (45) 11E7K1 (2) H1 (30) 10D7K (7) H1(35) 19G6K (15) H1 (44) 26C10K (20) H1 (51) 3A5K (6) H2 (34) 26H3K (14)H4 (43) 23H6K (11) H1 (39) 1A10K (1) H2 (29) 26H3K(c) (27) H4 (43) 26C7K(13) H1 (42) 25C9K (22) H1 (50) 24A2K (17) H1 (46) 26G2K (24) H1 (40)27D8K (8) H1 (36) 18A11K (9) H2 (37) 22F5 (19) H1 (49) 2F12K (4) H2 (32)26E9K (18) H1 (47) 1A10K(d) (28) H2 (53) 17C8K (21) H1 (52)

TABLE 3(b) Grouping of antibodies by lambda light chain, withcorresponding heavy chain Lambda chains Heavy chain (3 antibodies, nogroup consensus) (H1-H5) 14E4 (5) H1 (33) 27G8 (12) H3 (41) 27G8(a) (25)H3 (54) 27G8(b) (26) H3 (54) 19E6 (23) H5 (48)

CDR boundaries within the consensus sequences (which may vary, asdiscussed previously) were as follows: Kappa Group 1 CDR124-35,CDR251-57, CDR390-99; Kappa Group 2 CDR124-34, CDR251-56, CDR389-97;Kappa Group 3 CDR124-34, CDR250-56, CDR389-97; Heavy Chain Group 1CDR131-35, CDR250-66, CDR399-110; Heavy Chain Group 2 CDR131-35, CDR250-66, CDR399-107; Heavy Chain Group 3 CDR131-35, CDR250-66, CDR399-114;and Heavy Chain Group 4 CDR131-35, CDR250-66, CDR399-114.

EXAMPLE 3 Functional Assays

This example describes various assays that were used to characterize theantibodies.

HUT78 Adhesion Assay.

Coated plates (for example, Costar® 3368 96-well plates; CorningIncorporated Life Sciences, Lowell Mass.) are prepared by coating96-well plates overnight at 4° C. with 20 microG/mL MAdCAM-1 (or asimilar concentration of human IgG1 as a coating control) diluted inphosphate buffer pH9.0. The coating is removed and the plates areblocked with 100 microL of 3% BSA/PBS, incubated for 1 hr or more atroom temp. The plates are washed three times with Hank's balanced saltsolution (HBSS).

HUT78 cells (a human T cell lymphoma cell line that exhibits thefeatures of a mature T cell line with inducer/helper phenotype; ATCC TIB161), grown to confluency, are pelleted and washed 3× in HBSS, thenresuspended in HBSS at appropriate concentration to yield ˜30,000 cellsin 50 microL.

Antibodies to be tested are diluted to twice the final concentration,and then titrated 1:4 in calcium-free, magnesium free HBSS containing 1%BSA with 1 mM Mn²⁺. Fifty microL of antibody titration or control isadded to each well of a VEE bottom plate, followed by 50 microL of HUT78cells. The cells and antibodies are incubated at 4° C. for 30 minutes,then added to the coated plates and incubated at 37° C. for 40 minutes.Cells on coated plates are washed three times in room temperature HBSS,by flicking HBSS off between washes. The adherent cells arefreeze-thawed at −20 C followed by the addition of 100 microL ofCyQuant® dye/lysis buffer (a buffer used in a fluorescence-based cellquantification assays useful in high-throughput screening MolecularProbes®, Life Technologies Corporation, Carlsbad, Calif. The fluorescentsignal from each well is quantitated at 485 nm excitation and 530 nmemission, for example using a Tecan GENiosPro, a multi-label microplatereader (Tecan Group Ltd. Männedorf, Switzerland).

Human CD4+ Cell Adhesion Assay

Plates are coated with human MAdCAM-1-Fc or human IgG (3 microG/ml in 20mM phosphate buffer, pH 9.0, 130 mM NaCl), 100 microL/well, at 4° C.overnight then blocked with 200 microL/well blocking reagent (3% bovineserum albumen in PBS) at room temperature for at least two hours. Platesare then washed three times with adhesion buffer (30 mM HEPES, pH 7.4,120 mM NaCl, 1 mM MnCl₂, 10 g/ml Human IgG).

Serial dilutions of antibodies to be tested are prepared, and added tothe plate (35 microL/well); isolated CD4⁺ T cells (250,000 cells/35microL/well) are added and the plates are incubated at 4° C. for 2 hrs.After washing three times with adhesion buffer, plates are frozen at−20° C. overnight. Detection reagent (100 microL well CyQUANT® reagent;Life Technologies Corporation, Carlsbad, Calif.) is added and plates areincubated at 37° C. for 45 minutes. Results are determined by readingfluorescence at 485 nm excitation and 530 nm emission.

EC50 in Binding Human CD4+CD45RA-Memory T Cells

Human peripheral blood mononuclear cells (PBMC; fresh or frozen andthawed, for example in phosphate buffered saline with 2% FBS) are washedand resuspended in HEPES buffer (30 mM HEPES+140 nM NaCl) with 1% BSA,with or without 1 mM MnCl₂ (depending on the experiment; Mn²⁺ isnecessary for MAdCAM-1 binding) and plated into 96 well plates (10⁶cells/well). Cells are incubated with 10 microG/ml human IgG for 30minutes on ice to block nonspecific binding. Cells are then incubatedwith serial dilutions of biotinylated anti-alpha4beta7 antibodies in 96well plates for one hour on ice, followed by addition of 1:100 dilutionof streptavidin-phycoerythrin (PE; Jackson ImmunoResearch LaboratoriesInc., West Grove, Pa.), 4 microL CD3-Pacific Blue, CD4-PerCP-Cy5.5 andCD45RA-fluorescein isothiocyanate (FITC) (BD Biosciences, San JoseCalif.) for a final volume of 100 microL, and incubated for another houron ice. Cells were washed twice with HEPES buffer (with or withoutMnCl₂, correspondingly) and then fixed in 200 microL HEPES buffer plus0.5% paraformaldehyde (again, with or without MnCl₂, correspondingly).The percentage of positive alpha4beta7 antibody bindingCD4+CD45RA-memory T cells is determined using a fluorescence activatedcell sorter (FACS), for example, a BD™ LSR II benchtop flow cytometer(BD Biosciences, San Jose Calif.). EC50 is defined as the concentrationof alpha4beta7 antibody at which 50% of the alpha4beta7 sites onCD4CD45RA-memory cells are bound by the alpha4beta7 antibody.

IC50 in Blocking MAdCAM-1-Fc Binding to Human CD4+CD45RA-Memory T Cells.

PBMC (fresh or frozen as described previously) are washed andresuspended in HEPES buffer (30 mM HEPES+140 nM NaCl) with 1% BSA and 1mM MnCl. to a final concentration of 10⁷ cells/ml. Cells are blocked asdescribed previously; after blocking, cells are incubated with a serialdilution of anti-alpha4beta7 antibody (or appropriate control) in 96well plates for 30 minutes on ice, and then with 0.3 microG/mlbiotinylated MAdCAM-1-Fc protein for another one hour.

After two washes in HEPES buffer with 1 mM MnCl, cells are treated with1:100 dilution of streptavidin-PE, 4 microL CD3-Pacific Blue,CD4-PerCP-Cy5.5 and CD45RA-FITC as described previously, in a finalvolume of 100 microL. After one hour incubation on ice, cells are washedtwice with HEPES buffer with 1 mM MnCl and then fixed in 200 microLbuffer plus 0.5% paraformaldehyde. The percentage of positiveMAdCAM-1-Fc binding CD4+CD45RA-memory T cells is determined byfluorescence activated cell sorter (FACS) analysis, as describedpreviously. IC50 is defined as the concentration of alpha4beta7 antibodyat which MAdCAM-1-Fc binding to alpha4beta7 on CD4CD45RA-memory cells isinhibited by 50%.

Alpha4beta7 Induction by Retinoic Acid on Activated T Cells

Isolated human PBMC are activated by anti CD3 (plate bound, 5microG/ml), human IL-2 (20 ng/ml) in the presence or absence of retinoicacid (1000 nM) for 7 days. The activated cells are washed twice withstaining buffer (PBS plus 0.5% BSA and 1 mM MnCl) and incubated with 100microG/ml human Ig for 30 minutes to block non-specific binding. Thecells are first incubated in a serial dilution of anti-alpha4beta7antibodies for 30 minutes on ice, and then stained with 1 microG/mlbiotinylated MAdCAM-1-Fc for another 30 minutes. After twice washingwith staining buffer, cells are stained with Streptavidin-PE (1:1000)for 30 minutes. The cells are analyzed by fluorescence activated cellsorting, for example, with a FACSCalibur™ (BD Biosciences, San JoseCalif.). Cells prepared in this manner may be used for additionalexperiments such as competition assays.

Competition Assays

Alpha4beta7 antibodies were also examined for their ability to competewith other anti-alpha4beta7 and/or beta7 antibodies in binding to alpha4beta7 expressing cells by fluorometric microvolume assay technology orFMAT, substantially as described by Fiscella, et al., NatureBiotechnology 21:302-307; 2003. Briefly, cells expressing high levels ofalpha4beta7 are prepared, for example by transient co-transfection ofcells with nucleic acids encoding alpha4 and nucleic acids expressingbeta7. Stable cell lines are prepared in a similar manner, using cellsand protocols appropriate for stable transfection. Transfected cells arescreened, for example, by FACS, using antibodies to alpha4, antibodiesto beta7, and/or ligand (i.e., MAdCAM-1, for example, a MAdCAM-1-Fcfusion protein). Cells may undergo several cycles of sorting andselection to yield clonal cell lines with reproducible, elevated levelsof alpha4beta7 expression.

Binding to S250N Mutant

Antibodies were also evaluated for their ability to recognize the S250Npoint mutant in the beta7 chain, which is known to be critical for ACT-1binding (J. Immunol. 159:1497, 1997). 293 cells transientlyco-expressing alpha4beta7 having the S250N mutation in the beta chain(ref) are prepared in a similar manner as that previously described forpreparation of cells expressing high levels of alpha4beta7.

Briefly a total 1×10⁶ transfected cells for profile are collected usingcell-dissociation solution and spun down at 1000 rpm for 5 min. Thecells are then blocked with 0.5 ml blocking buffer (1% goat serum/PBS)for 30 min to 1 hr at 4° C. with shaking. For MAdCAM-1-Fc staining,cells are incubated for 1 hr at 4° C. with shaking in Mn²⁺ Buffer (1 mMMnCl₂ in 30 mM HEPES+1% goat serum). Cells are then spun down at 1000rpm/5 min, and 0.5 ml of fresh blocking buffer with along with 10microG/ml of primary antibody is added, followed by incubation for 30min to 1 hr at 4° C. with shaking. After two washes with 4 ml of coldPBS (each wash), secondary antibody (i.e., goat-antiIgG-Phycoerythrin-conjugated antibody; Southern Biotech, 1:250 dilutedor 0.1 microG/10⁶ cells) in 0.5 ml blocking buffer is added and cellsare incubated for 20-30 min at 4° C. Cells are washed one final timewith 4 mls of cold PBS, then resuspended in 0.5 ml of FACS buffer forprofile.Binding to Single Nucleotide Polymorphisms (SNPs)

For SNP analysis of the α4 subunit, α4 gene exons 1-28 from 90individuals (180 haploid genomes) representing different ethnic groupswere amplified by polymerase chain reaction (PCR) and subsequentlysequenced. Three candidate SNPs in the coding region of the α4 gene wereidentified, and one of the three resulted in an amino acid change(Arg878Gln). Similarly for the β7 subunit SNP analysis, the codingregion of β7 gene exons 2-15 from 90 individuals (180 haploid genomes)representing different ethnic groups were PCR amplified and subsequentlysequenced. Three SNPs were identified, and two of them resulted in aminoacid changes. The in-house SNP analysis data were compared withinformation in the NCBI database (NCBI: National Center forBiotechnology Information a division of the National Library of Medicine(NLM) at the National Institutes of Health (NIH)). Only the A/G mutationresulting in Gln878Arg in the α4 subunit occurs at high frequency—20% or30% in both in-house SNP and the public database respectively. The otherSNPs occur at low frequency. This information is summarized in Table 4below.

TABLE 4 Frequency of SNPs in human beta7 and alpha4 Alternative alleleAlternative allele frequency - NCBI frequency - in-house SNP databaseanalysis Location beta7 E97V NA A(0.989)/T(0.019) extracellular R213SC(0.975)/A(0.25) No observation extracellular G611E NA No observationextracellular G629S NA A(0.989)/T(0.019) extracellular H672Y NA Noobservation extracellular alpha4 V824A T(0.972)/ No observationextracellular C(0.028) Q878R A(0.648)/ A(0.783)/G(0.217) extracellularG(0.352) R1007S NA No observation intracellular

Point mutant constructs representing amino acid altering SNPs(a4b7(E97V); a4b7(R213S); a4b7(G629S; a4(V824A)b7; a4(Q878R)b7) in theextracellular domains of both alpha4 and beta7 were generated. Eachpoint mutant construct was transfected to 293 cells along with thewild-type partner expression construct. Transfected 293 cells were firststained with 1 microG/ml of human IgG or anti-alpha4beta7 antibody,washed with PBS, and the stained with phycoerytherin conjugatedsecondary antibody goat-anti human IgG. After washing with PBS, cellswere analyzed by fluorescence activated cell sorting, for example, witha FACSCalibur™ (BD Biosciences, San Jose Calif.). Fluorescence stainingintensity (geometric mean) for each antibody staining is indicated inTable 5 below.

TABLE 5 Binding to SNPs wt E97V R213S G629S V824A Q878R IgG 8 7 7 7 7 71A10 122 135 31 80 102 70 3A5 124 135 31 80 110 71 2F12 129 134 37 80112 73 18A11 92 105 30 63 82 56 22B2 97 108 58 65 85 58 26H3 93 106 4962 82 55 27G8 102 116 59 68 88 58 26G2 99 113 38 64 86 58 17C8 93 96 4958 74 51 19G6 94 108 46 59 67 46 25C9 96 106 33 59 77 51

These results indicated that all of the antibodies tested bound to theknown SNPs of alpha4beta7.

The activities of various alpha4beta7heterodimer specific antibodies inseveral different assays are compared in Table 6 below.

TABLE 6 Characterization of antibodies to alpha4beta7 HUT78 MAdCAM-1CD4+CD45RA- a4b7 Adhesion Competition Cell Binding (S250N) Antibody IC50(ng/ml) IC50 (ng/mL) EC50 (ng/mL) Binding 1A10 6.1 6.2 4.9 − 3A5 7.5 6.25.6 − 2F12 11.4 4.6 3.3 − 18A11 7.4 7.3 4.7 − 22B2 3.7 23.2 5.1 − 26H38.9 14.1 9.3 − 27G8 14.9 8.7 6.3 − 26G2 6.9 99.6 32.6 + 17C8 6.8 31.122.9 + 19G6 12.2 103.3 32.9 + 25C9 13.7 77.6 NA +

Soler et al. reported the binding specificity of a humanizedanti-alpha4beta7 antibody known as vedolizumab (J Pharmacol Exp Ther330:864; 2009). This antibody was reported to have an EC50 on memoryCD4+ T lymphocytes of 0.042 microgr/ml (42 ng/ml). Vedolizumab alsoinhibited the binding of soluble MAdCAM-1 to alpha4beta7hi memory Tcells with an 1050 of 0.034 microgr/ml (34 ng/ml). In contrast, many ofthe antibodies shown in Table 6 have an EC50 on memory T cells (i.e.,CD4+CD45RA-cells) of less than 10 ng/ml, and all of them have an EC50 ofless than 35 ng/ml (all also have an EC50 of greater than 0.1 ng/ml inthis assay). Additionally, several of the antibodies shown in Table 6demonstrated an IC50 in a MAdCAM competition assay of less than 10ng/ml, and many demonstrated an 1050 of less than 30 ng/ml (all alsoexhibited an IC50 of greater than 0.1 ng/ml in this assay). AlthoughSoler et al. made no mention of the ability of vedolizumab to bind anS250N mutant of alpha4beta7, the murine antibody ACT-1 from whichvedolizumab is derived is known to be unable to bind an S250N mutant(Tidswell et al., J Immunol 159:1497; 1997), and according to Soler etal., vedolizumab and ACT-1 exhibit the same antigen specificity. Thus,vedolizumab also does not bind the S250N mutant, in contrast to severalof the antibodies shown in Table 6.

EXAMPLE 4 Additional Analysis

Several representative antibodies with different properties in theafore-mentioned functional assays were chosen for additional analysis asdescribed below.

Binding Affinity to Human a4b7

To measure cell binding affinity of human anti-alpha4beta7antibodies, aKinetic Exclusion Assay which measures binding events in solution phasecan be used to calculate the equilibrium dissociation constant, K_(d).KinExA® Technology (Sapidyne Instruments, Boise, Id.) was used,substantially as described previously by Xie et. al. J. Imm, Methods304:1 (2005) and Rathanaswami et. al. Anal. Biochem. 373:52 (2008).Briefly, HUT78 cells expressing human alpha4beta7 were titrated 1 in 3from ˜50⁶ cells/mL to ˜400 cells/mL and then equilibrated with a finalconcentration of either 2 or 30 pM of mAb 2F12 or 18A11 and 30 or 500 pMfor 17C8 for 18 hours at 4° C. The free antibody remaining in thesupernatant at equilibrium was measured by KinExA® technology by passingthe supernatant over PMMA beads pre-coated with goat anti-human Fc anddetected with goat anti-human (H+L) Cy5 (substantially as described byRathanaswami et al. Biochem Biophys Research Commun: 1004 (2005). Theequilibrium dissociation constant (K_(d)) is obtained using KinExA®software by “n-curve analysis” which fits all of the given curves to asingle K_(d) value simultaneously (Rathanswami et al. 2005 and Xie etal., supra).; results are shown below in Table 7.

TABLE 7 Binding affinity of antibodies Ratio Antibody Kd (pM) (low Ab) %error Kd low Kd high 2F12 4.56 0.44 3.80 1.94 11.12 18A11 0.90 2.22 4.400.23 2.29 17C8 29.36 1.02 3.58 12.09 74.53

These antibodies demonstrated a Kd in a KinExA® assay greater than 0.05pM, but less than 80 pM, less than 15 pM, or less than 5 pM.

PK/PD Characteristics

A single-dose pharmacokinetic (PK) and pharmacodynamic (PD) study ofthree fully human anti-alpha4beta7 antibodies in male cynomologousmonkeys was conducted following intravenous (IV; 5 mg/kg) orsubcutaneous (SC; 0.5 or 5 mg/kg) administration. Similar initial PKexposure (C_(o); concentration at time zero) and distribution within thecentral circulation after 5 mg/kg IV was observed. After SCadministration, both C_(max) (maximum concentration in serum) and AUC(area under the concentration-time curve) exhibited dose-proportionalitywithin the 0.5-5 mg/kg SC dose range for all three antibodies. Theabsolute bioavailability after SC for the three tested antibodies rangedfrom 44 to 68%.

Free alpha4beta7 on T cells before and after antibody treatment werequantified by PE-conjugated anti-alpha4beta7 antibody 27G8. Thebackground level was controlled by staining with PE-conjugatedanti-alpha4beta7 antibody 27G8 in the presence of 10 mg/ml antibodybeing tested before and after antibody treatment. Fractional saturationwas determined by percentage of free alpha4beta7 sites in comparison tothe pre-treatment for each antibody. CD4-PerCP, CD99-APC and CD28-FITCwere used to distinguish naive, central memory and effector memorycells. Fractional saturation of alpha4beta7 on naive T cells was shownbecause of the variability and limited assay sensitivity in thecynomologous memory cell population. For 18A11 treatment, all threedosage groups remained saturated from day 1 to day 14. At day 29, allthree groups lost coverage. In both 2F12 and 17C8 treatment, loss oftarget coverage was observed on day 14 at the low dosage (0.5 mg/Kg)group.

In addition to free alpha4beta7 detection, target saturation was alsodetermined by staining with PE-conjugated anti-human antibody A35 in theabsence or presence of 10 mg/ml antibody. Total alpha4beta7 sites wereestimated with pre-incubation of samples with 10 mg/ml anti-alpha4beta7antibody followed by staining with anti-human antibody. Targetsaturation was determined by the percentage of total alpha4beta7 sitesoccupied by the anti-alpha4beta7 antibodies for each sample. The threefully human anti-alpha4beta7 antibodies demonstrated saturation ofalpha4beta7 that was maintained at mean of 81 to 100% within 14 daysafter 5 mg/kg IV.

PK/PD modeling was conducted on serum anti-alpha4beta7 antibodyconcentrations and corresponding alpha4beta7 receptor saturation datausing a direct E_(max) model. The model estimated PD parameters areE_(max)(maximum alpha4beta7 receptor saturation) of 92%, EC₅₀(anti-alpha4beta7 antibody concentration at which 50% of the E_(max) wasreached) of 52 ng/mL, and E₀ (initial alpha4beta7 receptor saturation)of 18%. All three antibodies exhibited potent in vivo PD effects onsaturating alpha4beta7 receptors with average t_(1/2) of ˜3-5 days incynomolgus monkeys.

What is claimed is:
 1. An isolated, alpha4beta7 heterodimer specificantigen binding protein having a heavy chain variable region comprisingCDR1, CDR2 and CDR3 from SEQ ID NO:37, and a light chain variable regioncomprising CDR1, CDR2 and CDR3 from SEQ ID NO:9.
 2. The heterodimerspecific antigen binding protein of claim 1, wherein the light chainvariable region is at least 90% identical to SEQ ID NO:9, and the heavychain variable region is at least 90% identical to SEQ ID NO:37.
 3. Theheterodimer specific antigen binding protein of claim 2, wherein thelight chain variable region comprises SEQ ID NO:9, and the heavy chainvariable region is selected from the group consisting of a polypeptidethat comprises SEQ ID NO:37 and a polypeptide that comprises SEQ IDNO:37 wherein the N-terminal amino acid is converted to pyroglutamicacid.
 4. A composition comprising the protein of claim 3 and aphysiologically acceptable diluent, excipient or carrier.
 5. Theisolated, alpha4beta7 heterodimer specific antigen binding protein ofclaim 3, which further comprises a light chain constant region and aheavy chain constant region.
 6. The isolated, alpha4beta7 heterodimerspecific antigen binding protein of claim 5 wherein the light chainconstant region is a kappa-type light chain constant region; and theheavy chain constant region is selected from the group consisting of:a′) a constant region from an IgD antibody; b′) a constant region froman IgE antibody; c′) a constant region from an IgM antibody; d′) aconstant region from an IgG1 antibody; e′) a constant region from anIgG2 antibody; f′) a constant region from an IgG3 antibody; g′) aconstant region from an IgG4 antibody; and h′) a constant region from anIgG4 antibody having at least one mutation in a hinge region thatalleviates a tendency to form intra-H chain disulfide bond.
 7. Acomposition comprising the protein of claim 6 and a physiologicallyacceptable diluent, excipient or carrier.
 8. The isolated, alpha4beta7heterodimer specific antigen binding protein of claim 5 wherein thelight chain constant region is selected from the group consisting of: a)a polypeptide comprising SEQ ID NO:70; b) a polypeptide at least 90%identical to SEQ ID NO:70; c) a polypeptide of a) which incorporates oneor more post-translational modifications; and d) a polypeptide having anamino acid sequence as set forth in SEQ ID NO:70 from which one, two,three, four or five N-terminal and/or C-terminal amino acids have beenremoved, and the heavy chain constant region is selected from the groupconsisting of: a′) a polypeptide comprising SEQ ID NO:72; b′) apolypeptide at least 90% identical to SEQ ID NO:72; c′) a polypeptide ofa′) which incorporates one or more post-translational modifications; andd′) a polypeptide having an amino acid sequence as set forth in SEQNO:72 from which one, two, three, four or five N-terminal and/orC-terminal amino acids have been removed.
 9. A composition comprisingthe protein of claim 8 and a physiologically acceptable diluent,excipient or carrier.
 10. A composition comprising the protein of claim5 and a physiologically acceptable diluent, excipient or carrier. 11.The isolated, alpha4beta7 heterodimer specific antigen binding proteinof claim 1, which further comprises a light chain constant region and aheavy chain constant region.
 12. The isolated, alpha4beta7 heterodimerspecific antigen binding protein of claim 11 wherein the light chainconstant region is a kappa-type light chain constant region; and theheavy chain constant region is selected from the group consisting of:a′) a constant region from an IgD antibody; b′) a constant region froman IgE antibody; c′) a constant region from an IgM antibody; d′) aconstant region from an IgG1 antibody; e′) a constant region from anIgG2 antibody; f′) a constant region from an IgG3 antibody; g′) aconstant region from an IgG4 antibody; and h′) a constant region from anIgG4 antibody having at least one mutation in a hinge region thatalleviates a tendency to form intra-H chain disulfide bond.
 13. Acomposition comprising protein of claim 12 and a physiologicallyacceptable diluent, excipient or carrier.
 14. The isolated, alpha4beta7heterodimer specific antigen binding protein of claim 11 wherein thelight chain constant region is selected from the group consisting of: a)a polypeptide comprising SEQ ID NO:70; b) a polypeptide at least 90%identical to SEQ ID NO:70; c) a polypeptide of a) which incorporates oneor more post-translational modifications; and d) a polypeptide having anamino acid sequence as set forth in SEQ ID NO:70 from which one, two,three, four or five N-terminal and/or C-terminal amino acids have beenremoved, and the heavy chain constant region is selected from the groupconsisting of: a′) a polypeptide comprising SEQ ID NO:72; b′) apolypeptide at least 90% identical to SEQ ID NO:72; c′) a polypeptide ofa′) which incorporates one or more post-translational modifications; andd′) a polypeptide having an amino acid sequence as set forth in SEQ IDNO:72 from which one, two, three, four or five N-terminal and/orC-terminal amino acids have been removed.
 15. A composition comprisingthe protein of claim 14 and a physiologically acceptable diluent,excipient or carrier.
 16. A composition comprising the protein of claim11 and a physiologically acceptable diluent, excipient or carrier.
 17. Acomposition comprising the protein of claim 2 and a physiologicallyacceptable diluent, excipient or carrier.
 18. The isolated, alpha4beta7heterodimer specific antigen binding protein of claim 2 which furthercomprises a light chain constant region and a heavy chain constantregion.
 19. The isolated, alpha4beta7 heterodimer specific antigenbinding protein of claim 18 wherein the light chain constant region is akappa-type light chain constant region; and the heavy chain constantregion is selected from the group consisting of: a′) a constant regionfrom an IgD antibody; b′) a constant region from an IgE antibody; c′) aconstant region from an IgM antibody; d′) a constant region from an IgG1antibody; e′) a constant region from an IgG2 antibody; f′) a constantregion from an IgG3 antibody; g′) a constant region from an IgG4antibody; and h′) a constant region from an IgG4 antibody having atleast one mutation in a hinge region that alleviates a tendency to formintra-H chain disulfide bond.
 20. A composition comprising the proteinof claim 19 and a physiologically acceptable diluent, excipient orcarrier.
 21. The isolated, alpha4beta7 heterodimer specific antigenbinding protein of claim 18 wherein the light chain constant region isselected from the group consisting of: a) a polypeptide comprising SEQID NO:70; b) a polypeptide at least 90% identical to SEQ ID NO:70; c) apolypeptide of a) which incorporates one or more post-translationalmodifications; and d) a polypeptide having an amino acid sequence as setforth in SEQ ID NO:70 from which one, two, three, four or fiveN-terminal and/or C-terminal amino acids have been removed, and theheavy chain constant region is selected from the group consisting of:a′) a polypeptide comprising SEQ ID NO:72; b′) a polypeptide at least90% identical to SEQ ID NO:72; c′) a polypeptide of a′) whichincorporates one or more post-translational modifications; and d′) apolypeptide having an amino acid sequence as set forth in SEQ ID NO:72from which one, two, three, four or five N-terminal and/or C-terminalamino acids have been removed.
 22. A composition comprising the proteinof claim 21 and a physiologically acceptable diluent, excipient orcarrier.
 23. A composition comprising the protein of claim 18 and aphysiologically acceptable diluent, excipient or carrier.
 24. Acomposition comprising the protein of claim 1 and a physiologicallyacceptable diluent, excipient or carrier.
 25. An isolated, alpha4beta7heterodimer specific antigen binding protein having a heavy chainvariable region and a light chain variable region, wherein the heavychain variable region is encoded by a nucleic acid that encodes SEQ IDNO:37, and the light chain variable region is encoded by a nucleic acidthat encodes SEQ ID NO:9.
 26. A composition comprising the protein ofclaim 25 and a physiologically acceptable diluent, excipient or carrier.27. The isolated, alpha4beta7 heterodimer specific antigen bindingprotein of claim 25 wherein the nucleic acid that encodes the heavychain variable region comprises SEQ ID NO:64, and the nucleic acid thatencodes the light chain variable region comprises SEQ ID NO:63.
 28. Acomposition comprising the protein of claim 27 and a physiologicallyacceptable diluent, excipient or carrier.
 29. The isolated, alpha4beta7heterodimer specific antigen binding protein of claim 27, which furthercomprises a light chain constant region and a heavy china constantregion, wherein the light chain constant region is encoded by a nucleicacid that encodes SEQ ID NO:70 and the heavy chain constant region isencoded by a nucleic acid that encodes SEQ ID NO:72.
 30. The isolated,alpha4beta7 heterodimer specific antigen binding protein of claim 29wherein the heavy chain constant region is encoded by a nucleic acidthat comprises SEQ NO:69, and the light chain constant region is encodedby a nucleic acid that comprises SEQ ID NO:71.
 31. A compositioncomprising the protein of claim 30 and a physiologically acceptablediluent, excipient or carrier.
 32. A composition comprising the proteinof claim 29 and a physiologically acceptable diluent, excipient orcarrier.
 33. The isolated, alpha4beta7 heterodimer specific antigenbinding protein of claim 25, which further comprises a light chainconstant region and a heavy china constant region, wherein the lightchain constant region is encoded by a nucleic acid that encodes SEQ IDNO:70 and the heavy chain constant region is encoded by a nucleic acidthat encodes SEQ ID NO:72.
 34. The isolated, alpha4beta7 heterodimerspecific antigen binding protein of claim 33 wherein the heavy chainconstant region is encoded by a nucleic acid that comprises SEQ IDNO:69, and the light chain constant region is encoded by a nucleic acidthat comprises SEQ ID NO:71.
 35. A composition comprising the protein ofclaim 34 and a physiologically acceptable diluent, excipient or carrier.36. A composition comprising the protein of claim 33 and aphysiologically acceptable diluent, excipient or carrier.